Evaluation of the selectivity and sensitivity of isoform ... · Evaluation of the selectivity and sensitivity of isoform- and mutation-specific RAS antibodies Andrew M. Waters,1,2

SC I ENCE S I GNAL ING | R E S EARCH RE SOURCE

TECHN IQUES

1Lineberger Comprehensive Cancer Center, University of North Carolina at ChapelHill, Chapel Hill, NC 27599, USA. 2National Cancer Institute RAS Initiative, CancerResearch Technology Program, Frederick National Laboratory for Cancer Re-search, Leidos Biomedical Research Inc., Frederick, MD 21702, USA. 3Departmentof Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, NC27599, USA. 4Greehey Children’s Cancer Research Institute, University of TexasHealth Science Center at San Antonio, San Antonio, TX 78229, USA. 5Departmentof Molecular Oncology, BC Cancer Research Centre, Vancouver, British ColumbiaV5Z 1L3, Canada. 6UCSF Helen Diller Family Comprehensive Cancer Center,School of Medicine, University of California at San Francisco (UCSF), San Francisco,CA 94143, USA. 7Department of Radiation Oncology, University of North Carolinaat Chapel Hill, Chapel Hill, NC 27599, USA. 8Perlmutter Cancer Institute, New YorkUniversity School of Medicine, New York, NY 10016, USA.*Corresponding author. Email: [email protected]

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Evaluation of the selectivity and sensitivity ofisoform- and mutation-specific RAS antibodiesAndrew M. Waters,1,2 Irem Ozkan-Dagliyan,3 Angelina V. Vaseva,1,4 Nicole Fer,2

Leslie A. Strathern,2 G. Aaron Hobbs,1 Basile Tessier-Cloutier,5 William K. Gillette,2 Rachel Bagni,2

Gordon R. Whiteley,2 James L. Hartley,2 Frank McCormick,2,6 Adrienne D. Cox,1,3,7

Peter J. Houghton,4 David G. Huntsman,5 Mark R. Philips,8 Channing J. Der1,3*

There is intense interest in developing therapeutic strategies for RAS proteins, the most frequently mutated oncopro-tein family in cancer. Development of effective anti-RAS therapies will be aided by the greater appreciation of RASisoform–specific differences in signaling events that support neoplastic cell growth. However, critical issues thatrequire resolution to facilitate the success of these efforts remain. In particular, the use of well-validated anti-RASantibodies is essential for accurate interpretation of experimental data. We evaluated 22 commercially availableanti-RAS antibodies with a set of distinct reagents and cell lines for their specificity and selectivity in recognizingthe intended RAS isoforms andmutants. Reliability varied substantially. For example, we found that some pan- orisoform-selective anti-RAS antibodies did not adequately recognize their intended target or showedgreater selectivityfor another; somewere valid for detecting G12D and G12Vmutant RAS proteins inWestern blotting, but none werevalid for immunofluorescence or immunohistochemical analyses; and some antibodies recognized nonspecificbands in lysates from “Rasless” cells expressing the oncoprotein BRAFV600E. Using our validated antibodies, weidentified RAS isoform–specific siRNAs and shRNAs. Our results may help to ensure the accurate interpretation offuture RAS studies.

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INTRODUCTIONThe three RAS genes (HRAS, KRAS, and NRAS) comprise the mostfrequently mutated oncogene family in cancer, with an overall 27%missense mutation frequency in all human cancers (COSMIC v80)(1–3).RASmutation frequency is highly tissue context–dependent, withhighest frequencies observed in lung, colorectal, and pancreatic ductaladenocarcinoma (PDAC).

RAS genes encode four structurally and biochemically highly related188 to 189 amino acidproteins (4).KRAS encodes twoproteins,KRAS4Aand KRAS4B, owing to alternative fourth exon utilization (5–8). RASproteins exhibit 82 to 90% overall amino acid sequence identity (9)and 93 to 99% sequence identity within the N-terminal G domain (resi-dues 1 to 164) (10). In sequences involved in interactionwith the guaninenucleotide exchange factors and guanosine triphosphatase (GTPase)–activatingproteins that regulate guanosinediphosphate (GDP)–guanosinetriphosphate (GTP) cycling and in activation of downstream effectors(for example, RAF), this region is invariant. In contrast, RAS proteinsshow only 8% sequence identity in the C-terminal hypervariable region(HVR) (residues 165 to 184/185). RAS intracellular trafficking andmembrane interactions are determined by the divergentHVR andCaaxtetrapeptide motif–signaled posttranslational lipid modifications (pre-nylation andmethylation), contributing toRAS isoform–distinct biolog-

ical functions. One issue now recognized, yet still poorly understood,is that the four RAS protein isoforms, despite their near-identicalstructure and biochemistry, serve distinct roles as cancer drivers (3, 9).

The three RAS genes are not mutated at comparable frequencies inhuman cancers. KRAS is by far the most commonly mutated (84%),followed by NRAS (12%) and HRAS (4%) (1). In addition, there aretissue-specific preferences for the specificRAS genemutated. For exam-ple, KRAS is exclusively the RAS isoformmutated in PDAC, whereasNRAS is the predominant (94%) isoform mutated in cutaneous mela-noma, andHRASmutations are favored (86%) in head and neck squa-mous cell carcinoma. Limited evidence that these frequencies reflecttissue-specific functions comes from mouse models of cancer. For ex-ample, KrasG12D, but notNrasG12D, promoted colon cancer develop-ment in Apc-deficient mice (11). Expression of activated KrasG12V andHrasG12V under control of the same regulatory sequences resulted indifferent types of tumors (12).

Cancer-associated RAS genes containmissensemutations that re-sult in single amino acid substitutions; 99% are found at one of threemutational hotspots, glycine-12 (G12), glycine-13 (G13), or glutamine-61 (Q61) (1, 9, 13). G12mutations comprise 83% of allKRASmutations,followed by G13 mutations (14%), whereas Q61 mutations are rare(2%). In striking contrast, Q61 is the predominant mutational hotspotin NRAS (62%), followed by G12 (23%), and then G13 (12%). Thesefrequencies suggest that G12 and Q61 mutations may have distinctconsequences for different RAS isoforms. That NrasQ61R but notNrasG12D potently caused melanoma development in Ink4a-deficientmice supports this possibility (14). Finally, although there are six pos-sible single-basemissensemutations at each codon, the frequencieswithwhich they occur are not uniform. It has also become evident that RASmutant proteins exhibit distinct biochemical deficiencies (15, 16). Anemerging premise in the field is that the different RAS mutations arenot created equal and that the identification of mutation-selective dif-ferences will identify unique vulnerabilities that can then be exploitedto developmutation-selective therapies (17). The development of RAS

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inhibitors that are selective for one RAS mutation, G12C, representsthis shift in the field (18, 19).

Antibodies are essential reagents in biomedical research, but poor-quality antibodies are a major cause of the “reproducibility crisis” inunderstanding protein function and in the pathway to new drugs(20–22). This crisis, and the related need for better characterized andvalidated antibodies, is exemplified by a report that 47 of 53 landmarkcancer research papers from the early 2000s could not be replicated(23). It has been estimated that fewer than half of the 6000 commerciallyavailable antibodies recognize their advertised targets (21) and that$800million annually is spent on poor-quality antibodies ($350millionin the United States alone) (22). There are also numerous examples ofantibody problems slowing medical progress. In 2009, a researchgroup secured federal funding to stratify melanoma patient treatmentregimens based on quantitative immunofluorescence (IF) staining ofbiopsy samples (24). However, they were forced to abandon this projectbecause subsequent lots of antibodies from the same source no longergenerated the same results on the same tumor samples (20). Further-more, another research group spent 2 years, thousands of patientsamples, and $500,000 before realizing that the antibody they had beenusing for early detection of pancreatic cancerwas recognizing thewrongprotein (20), an astounding disappointment for pancreatic cancerpatients, whose 5-year survival rate is an abysmal 8% (25). Nevertheless,despite extensive efforts of organizations such as the InternationalWorking Group on Antibody Validation, two International AntibodyValidation Meetings, and crowd-funded scientific websites such aswww.antybuddy.com (26, 27), the quality of commercially availableantibodies and their degree of validation remain decidedly mixed.

With renewed interest in RAS and with new investigators enteringthe field (28, 29), we believe that now is an ideal time to revisit validationof RAS antibodies. To ensure the accuracy of our own studies and asa service to the field, we analyzed 22 commercially available RASantibodies for their specificity and sensitivity in recognizing individualRAS isoforms. Our analyses demonstrate that the properties of manyof these antibodies have not been accurately established. Our resultssupport the likelihood that the conclusions from some RAS researchhave been compromised by misinterpretation of data because of faultyinformation regarding RAS antibody properties. Moving forward,we hope that our validation of antibodies for specific applications willfacilitate greater reliability in future RAS studies.

RESULTSEvaluation of RAS isoform selectivity against recombinantRAS proteinsWepurified unprocessedwild-type full-length humanKRAS4B,HRAS,andNRASproteins fromEscherichia coli. In addition, both unprocessedand processed (farnesylated, proteolyzed, and methylated) KRAS4Band KRAS4A were purified from insect cells, as we described (30).Three concentrations of each purified RAS protein were resolved bySDS–polyacrylamide gel electrophoresis (PAGE) to verify size andpurity (Fig. 1A). KRAS bands appear slightly more intense across theblot, likely because of their lysine-rich C-terminal HVR sequencesdetected by the negatively charged Coomassie staining method. Nota-bly, unprocessed KRAS4A protein from insect cells purified as two spe-cies: aminority of the full-length protein and amajor species that, basedonmass spectrometry results, lacks the last five amino acids. Otherwise,RAS proteins were highly pure (>95%) and migrated at the expectedmolecular weight (MW) for full-length proteins.


We analyzed a panel of 22 commercially available RAS antibodies(Table 1) that includes those most widely cited in the literature. Alsoincluded in this panel were four monoclonal antibodies generatedagainst human KRAS4B protein by the National Cancer Institute(NCI) RAS Initiative at the Frederick National Laboratory for CancerResearch (FNLCR) that have not yet been used in any published studies.We interrogated each antibody for RAS isoform selectivity and sensitiv-ity and asked whether Caax-signaled processing influenced recognitionof KRAS proteins (Fig. 1, B to D). Most antibodies recognized theirintended target, but there were some exceptions.

No antibody described as having pan-RAS selectivity uniformlyrecognized all RAS isoforms. Ras10 (05-516) showed fairly equivalentrecognition of all RAS isoforms and was not influenced by KRAS4Bprocessing. However, another pan-RAS antibody (05-1072) was moreselective for HRAS and for unprocessed KRAS4A than for other RASproteins. Three of the four FNLCR-generated antibodies recognizedall the RAS proteins, although the immunogen used was recombinantfull-length KRAS4B (Fig. 1B). Notably, the FNLCR CPTC-KRAS4B-2 antibody showed pan-RAS sensitivity that was superior to any ofthe commercially available pan-RAS antibodies we tested.

Two HRAS-specific (SC-520 and 18295-1-AP) and two NRAS-specific (SC-31 and SC-519) antibodies showed strong selectivity fortheir intended targets, with no recognition of the other RAS isoforms(Fig. 1C).However, one antibody intended to beNRAS-specific (10724-1-AP) only weakly recognized NRAS at the highest protein abundanceand instead more effectively recognized other RAS isoforms.

We evaluated three commercially available KRAS4A/4B antibodies(Fig. 1D). Two antibodies (SC-30 and OP24) recognized unprocessedand processed versions of both KRAS isoforms, whereas another(WH0003845M1) showed strong selectivity for KRAS4B with a higheraffinity for unprocessed protein and did not recognize KRAS4A. Weevaluated three KRAS4A-specific antibodies [16156-1-AP, SC-522(C-17), and ABC1422], none of which recognized KRAS4B (Fig. 1D).However, all three showed significantly stronger recognition of un-processed than processed KRAS4A. This is not entirely surprising,given that the Caax-signaled posttranslational modifications are imme-diately adjacent to the major sequence differences between KRAS4Aand KRAS4B and may therefore disrupt epitope recognition of anti-bodies generated against purified bacterial proteins that lack suchmodi-fications. The two KRAS4B-selective antibodies recognized purifiedKRAS4B but did not recognize KRAS4A. One of these (16155-1-AP)recognized both unprocessed and processed KRAS4B proteins withcomparable signal strength (Fig. 1D). In contrast, the other (SC-521)recognized only unprocessed KRAS4B protein, although the antibodywas generated against the human KRAS4B C terminus. ProcessedKRAS4B may not be recognized because the antibody epitope overlapswith the cleaved Caax fragment that is removed during processing.

Evaluation of RAS isoform selectivity in lysates ofengineered mouse cellsWe next investigated how well the antibodies performed when probingcell lysates from cells deficient in any Ras genes [henceforth called Ras-less mouse embryonic fibroblasts (MEFs)] and rescued by BRAFV600E

or exogenous expression of a single RAS isoform. This approach tookadvantage of an innovative cell model developed by Drosten et al. (31).Rasless MEFs are derived from Hras−/−/Nras−/− mice harboring aconditional Kras allele that is deleted upon ectopic expression of Crerecombinase (31). UponKras deletion, theseMEFs lack all Ras isoformsand become quiescent upon loss of all endogenous Ras activity. As was

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shown previously (31), we also determined that proliferation can berescued by ectopic expression of activated BRAFV600E or of wild-typeversions of humanKRAS4A, KRAS4B, HRAS, or NRAS.We establishedMEF cell lines using clonal populations from cultures infected with len-tivirus expression vectors encoding each gene. Therefore, total RASprotein across the cell lines is comparable but most likely not equiv-alent (Fig. 2).

The results obtained with the three commercial pan-RAS antibodiesin cell lines largely mirrored those using recombinant RAS proteins(Fig. 2A). Antibody 05-516 readily detected all RAS isoforms, albeitmore weakly for KRAS4B. However, it also weakly detected a band inthe Rasless MEFs expressing BRAFV600E that comigrated with HRASandNRAS, likely aRAS-related protein.As described above, three of thefour FNLCR-generated anti-KRAS4B antibodies display pan-RAS spec-ificity, with CPTC–KRAS4B-2 showing stronger and equivalent recog-nition of all RAS isoforms, no detection of any proteins in the ~21-kDarange in the Rasless MEFs expressing BRAFV600E, and faint recognitionof other high-MW cellular proteins (Fig. 2B). CPTC–KRAS4B-3displayed similar pan-RAS reactivity and may better recognize NRAS.However, this antibody also weakly recognized a band in Rasless MEFsexpressing BRAFV600E that migrates just above all the RAS isoforms;depending on the properties of the SDS-PAGE used to resolve cell ly-sates, thisminor bandmay ormay not be a concern inWestern blottingapplications.

Although both HRAS antibodies showed high specificity for theHRAS isoform, one (18295-1-AP) recognized few other cellular pro-


teins, whereas the second (SC-520) also recognized numerous others(Fig. 2C). The NRAS antibody SC-31 showed high specificity forNRAS and no recognition of any ~21-kDa bands in Rasless MEFsexpressing BRAFV600E (Fig. 2D).

Of three putative KRAS4A/4B antibodies evaluated, both SC-30 andOP24 recognized both KRAS4A and KRAS4B, and WH0003845M1was highly selective for KRAS4B (Fig. 2E). However, on the basis ofthe much stronger recognition of recombinant KRAS4A protein bySC-30 and OP24 (Fig. 1D), we suspect that neither antibody wouldprovide an accurate determination of how expression of the twoKRAS isoforms at the RNA level is related to isoform expression atthe protein level.

One KRAS4A-specific antibody (16156-1-AP) appeared to recog-nize KRAS4A but additionally recognized a closely comigrating bandin Rasless MEFs expressing BRAFV600E (Fig. 2F). In contrast, two otherKRAS4A-specific antibodies selectively recognized KRAS4A, but notKRAS4B, with no detection of any ~21-kDa bands in Rasless MEFsexpressing BRAFV600E (Fig. 2F). Although both KRAS4B antibodiesspecifically detected purified recombinant proteins (Fig. 1D), neitherdetected KRAS4B in cell lysates but strongly recognized numerousnonspecific proteins (Fig. 2G). Instead, our data indicate that althoughWH0003845M1 is described as capable of detecting both KRAS4A andKRAS4B, it is the best available reagent for selective detection ofKRAS4B. We conclude that there are many experimental conditionsunder which even antibodies commonly used in the literature maylead to erroneous interpretations.

Fig. 1. Selectivity of RAS antibody detection of purified RAS proteins. Three different concentrations of E. coli (Ec)– or insect cell–expressed unprocessed (UP) orprocessed (P) purified RAS proteins were resolved by SDS-PAGE and (A) stained with Coomassie blue to show protein loading, or subjected to Western blot analysisusing a panel of RAS antibodies: (B) pan-RAS, (C) HRAS- or NRAS-selective, and (D) “pan-KRAS” KRAS4A/4B-, or KRAS4A- or KRAS4B-selective.

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Table 1. List of RAS antibodies and manufacturer information. mAb, monoclonal antibody; GST, glutathione S-transferase; FL, full-length; DSHB, Devel-opmental Studies Hybridoma Bank; NA, not applicable.

Wa

Isoform

ters et a

Catalog no.

l., Sci. Signal. 10,

Lot no.

eaao3332 (

Vendor C

2017) 26 September 2

lonality

017

Cloneno.

Host
Speciesreactivity*
Applications†
Immunogen/epitope
H, K, N
05-1072 2557627 Millipore mAb 9A11.2 M ouse H, M, R WB, IHC Recombinant GST-FL HRAS
H, K, N
05-516 2673037 Millipore mAb Ras10 M ouse H, M, R W B, ELISA, FC, ICC,IHC, IP
Recombinant RAS

H, K, N
3339 5 Cell SignalingTechnology
mAb
27H5 R abbit H, M, R WB N-terminal KRAS peptide
K
CPTC–KRAS4B-1‡
1
FNLCR/DSHB mAb 2D2 M ouse H WB Recombinant FL KRAS4B
K
CPTC–KRAS4B-2‡
1
FNLCR/DSHB mAb 4E8 M ouse H WB Recombinant FL KRAS4B
Do
K
wn

CPTC–KRAS4B-3‡

1
FNLCR/DSHB mAb 4H2 M ouse H WB Recombinant FL KRAS4B
l
oad K
ed

CPTC–KRAS4B-4‡

1
FNLCR/DSHB mAb 6C10 M ouse H WB Recombinant FL KRAS4B from K W H0003845M1 F A191-S2 Sigma-Aldrich mAb
3B10-2F2

M
ouse H WB, indirectELISA, IF
Recombinant GST-FL KRAS4B

h
ttp:/
K
SC-30 K0315
/s

Santa CruzBiotechnology

mAb
F234 M ouse H, M, R WB, IP, IF Recombinant rat KRAS4A(amino acids 54 to 189)
tk
e.s K OP24 D 00175459 Millipore mAb 234-4.2 M ouse H, M, R
cie

WB, frozensections, IP

Recombinant rat KRAS4A(amino acids 54 to 189)

n
ce H SC-520 L3015
ma


P
olyclonal NA R abbit H, M, R WB, IP, IF, ELISA Human HRAS C-terminalpeptide
g
.or H 18295-1-AP 22367 Proteintech P olyclonal NA R abbit H, M
g/

WB, ELISA, IF,IHC, FC

Peptide

o
n F N SC-31 L0915
eb


mAb
F155 M ouse H, M, R WB, IP, IF, IHC Recombinant RAS
ruar
N SC-519 (C-20) E0515
y


P
olyclonal NA R abbit H, M, R W B, IP, IF, IHC, ELISA Human NRAS C-terminalepitope
1
6, 2 N 10724-1-AP 17213 Proteintech P olyclonal NA R abbit H, M, R
02

WB, ELISA, IHC,FC, IF

Recombinant GST-FL humanNRAS protein

1

K4A
16156-1-AP 26025 Proteintech P olyclonal NA R abbit H WB, ELISA, IHC,IF, FC
Peptide

K4A
SC-522 (C-17) H2815 Santa CruzBiotechnology
P
olyclonal NA R abbit H, M, R WB, IP, IF, ELISA Human KRAS4A C-terminalepitope
K4A
ABC1442 Q 2737518 Millipore P olyclonal NA R abbit H WB KLH-KRAS4A peptide
K4B
SC-521 (C-19) I2315 Santa CruzBiotechnology
P
olyclonal NA R abbit H, M, R WB, IP, IF, IHC Human KRAS4B C-terminalepitope
K4B
16155-1-AP 15812 Proteintech P olyclonal NA R abbit H, M WB, ELISA, IHC,IF, FC
Peptide

Mutant-specificantibodies

G12D
14429S 1 Cell SignalingTechnology
mAb
D8H7 R abbit H WB RAS G12D mutant peptide
G12V
14412S 1 Cell SignalingTechnology
mAb
D2H12 R abbit H WB RAS G12V mutant peptide
*Species: H, human; M, mouse; R, rat. †Applications: IF, immunofluorescence; IHC, immunohistochemistry; IP, immunoprecipitation; FC, flow cytometry; ICC,immunocytochemistry; WB, Western blotting; ELISA, enzyme-linked immunosorbent assay. ‡Hybridoma.

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RAS isoform detection in a human cell lineOur analyses in Rasless MEFs assessed detection of exogenouslyexpressed human RAS and also revealed highly variable nonspecificrecognition ofmouse proteins. To assess detection of endogenous humanRAS proteins and to evaluate nonspecific reactivity in human cells,we also evaluated several antibodies using lysates of human embryonickidney (HEK) 293T cells (Fig. 3A). Two pan-RAS antibodies, 05-1072and 05-516, recognized all RAS isoforms but also recognized otherbands. The FNLCR antibody (CPTC–KRAS4B-2) that showed strongpan-RAS reactivity in RaslessMEFs expressing any RAS isoformwithoutadditional ~21-kDa bands in Rasless MEFs expressing BRAFV600E

(Fig. 2B) also showed strong recognition of human RAS proteins andminimal recognition of nonspecific human proteins.

Neither HRAS-specific antibody detected any ~21-kDa band inHEK293T cells, indicating that these cells do not express significantendogenous HRAS. The NRAS-selective antibody SC-31 strongly re-cognized endogenous NRAS, with essentially no nonspecific reactivity.

Collectively, our results using the three commercially availableKRAS4A/4B antibodies in all threemodel systems enable us to concludethat HEK293T cells express only KRAS4B and that WH0003845M1is superior to the two commercial KRAS4B-selective antibodies inrecognizing endogenous human KRAS4B. Similarly, despite the strongdetection by 16156-1-AP of a ~21-kDa band in HEK293T cells, we


conclude, based on our results using this antibody in RaslessMEFs, thatthis band likely represents detection of RAS-related proteins (such asRRAS isoforms). Finally, the lack of detection of any ~21-kDa bandin HEK293T cells by the KRAS4A-specific antibodies SC-522 andABC1442, coupled with the results from Rasless MEFs, validates bothas excellent reagents for selective detection by Western blotting ofendogenous humanKRAS4Abut notKRAS4Bprotein.We suggest thatsuch parallel analyses, using purified human RAS proteins, RaslessMEFs, and human cells, can more definitively identify the true specific-ity of RAS antibodies than any one system alone.

Use of RNA interference for antibody validation in humantumor cell linesOther experimental strategies that assist in the validation of the RASisoform selectivity of antibodies use genetic knockdown using shorthairpin RNA (shRNA) and small interfering RNA (siRNA) to selec-tively target each RAS gene. Currently, no shRNA has been reportedthat can selectively silence each KRAS splice variant. Using a panel ofhuman non–small cell lung carcinoma (NSCLC) cell lines, we first sta-bly infected each line with lentivirus vectors encoding a nonspecificsequence or two distinct targeted sequences (table S1) for humanHRAS, NRAS, or KRAS (Fig. 3B). Western blots on the resulting celllysates with our isoform-selective KRAS4A/4B andNRAS antibodies

Fig. 2. RAS antibodies detect both RAS and alsobackground bands and nonspecific 21-kDa bandsin lysates of rescued Rasless MEFs.Western blot analy-ses were performed with (A and B) pan-RAS, (C) HRAS-selective, (D) NRAS-selective, (E) KRAS4A/B-selective, (F)KRAS4A-selective, or (G) KRAS4B-selective antibodies ontotal cell lysates from Rasless MEFs ectopically expressingthe indicated proteins. Vinculin (A to F) or backgroundnonspecific bands (G) served as a loading control. Full(uncut) blots, including MagicMark XP size standards inthe left lane of each panel, are shown to reveal degreeof nonspecific protein recognition.

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validated in RaslessMEFs expressing KRAS orNRAS (OP24 and SC-31, respectively). For comparison, we included an HRAS-selectiveantibody, SC-520, which failed in our validation experiments, to illus-trate complications that may arise upon recognition of closely migrat-ing nonspecific reactivity.

The protein abundances detected by the KRAS4A/4B and NRASantibodies were strongly reduced in cells stably expressing shRNAtargeting KRAS and NRAS, respectively (Fig. 3B). In contrast, ~21-kDabands were not reduced in cells stably expressing shRNA targetingHRAS and probed with the SC-520 antibody, suggesting that theseHRAS-targeted shRNAs were not effective in silencing HRAS ex-pression. However, the same analyses with a well-validated secondHRAS-selective antibody, 18295-1-AP, did show strong suppressionof HRAS protein levels (Fig. 3, C and D). Thus, the apparent detection


of HRAS protein by SC-520 likely reflects instead its strong detection ofnon-RAS proteins thatmigrate nearHRAS (Fig. 2C). Table 2 providesa list of the isoform-specific, pan-RAS, and pan-KRAS (KRAS4A/4B)antibodies that we have validated forWestern blot applications, basedon the above experiments.

With our validated antibodies, we could initially identify shRNAsthat were selective for human KRAS or NRAS, without displayingoff-target effects on other RAS isoforms. However, both of the orig-inal shRNAs targeting HRAS had isoform-specific off-target effects(Fig. 3C). To better identify an effective and selective HRAS shRNA,we further infected the panel ofNSCLC lines with five additionalHRASshRNAs (Fig. 3D) and validated shRNAs that selectively suppressedHRAS. Our results using isoform-specific shRNAs are summarizedin table S1.

Fig. 3. RAS antibodies detect both endogenous RAS and nonspecific background bands in human cell lysates. (A) HEK293T cell lysates were probed by Westernblotting with the indicated RAS antibodies. Full (uncut) blots, including MW markers in the right lane of each panel, are shown to reveal the degree of nonspecificprotein recognition. (B and C) To determine isoform selectivity of shRNAs, we infected human NSCLC cell lines with lentivirus vectors encoding shRNAs targeting theindicated RAS genes or a nonspecific control (NS). Cell lysates were probed by Western blotting with the indicated RAS antibodies. Vinculin served as a loading control.(D) To identify HRAS-selective shRNAs, we infected seven HRAS-targeting constructs into NSCLC cells and probed them with the indicated RAS antibodies. (E and F) Todetermine isoform selectivity of siRNAs, we transfected rhabdomyosarcoma cell lines with siRNAs targeting the indicated RAS genes or a mismatch control (MM). Celllysates were probed by Western blotting with the indicated RAS antibodies. b-Actin served as a loading control.

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Similarly, we evaluated isoform specificity of siRNAs used to targetdifferent RAS isoforms in two RAS mutant rhabdomyosarcoma celllines. The protein levels detected by the specific antibodies were reducedin cells transiently transfected with the appropriate siRNAs (Fig. 3E). Inthis instance, HRAS knockdown is clearly visible with the SC-520 anti-body, suggesting that the nonspecific bands that comigrate with HRASin NSCLC lines are not expressed in rhabdomyosarcoma lines (Fig. 3,E and F), stressing that our antibody validations are context- and celltype–dependent. Notably, NRAS protein expression is reduced withKRAS siRNA1, HRAS siRNA2 decreases NRAS expression, and NRASsiRNA2and siRNA3reduceHRASexpression.Asdonewith the shRNAs,we further transfected these cells with additional KRAS, HRAS, andNRAS siRNAs and validated those that selectively suppress the correctisoformwithout targeting other RAS isoforms.Our results using isoform-specific siRNAs, as well as a list of all siRNA and shRNA binding sites,are summarized in the Supplementary Materials (table S2 and fig. S1,respectively).

Analysis of RAS G12D and G12Vmutation-specific antibodiesWe also tested two commercially available G12 mutant–specific RASantibodies for their ability to recognize ectopically expressed mutanthuman KRAS4B in rescued Rasless MEFs expressing different mutantKRASproteins and endogenousmutantKRAS in humanKRASmutantcell lines (Fig. 4). These antibodies were very specific for the indicatedmutant proteins. No background bands were detected in any of thelysates, even at extended exposure times. Notably, our data indicatethat KRAS protein levels in Rasless MEFs expressing different mutantKRAS proteins are comparable to endogenous levels in the establishedcancer cell lines. However, although very specific for their targets, thesemutation-specific antibodies were considerably less sensitive than mostof the isoform-specific antibodies. We included only KRAS mutant celllines along with our controls in these experiments, but these antibodiesare also advertised to detect the G12D and G12V mutant forms ofHRAS and NRAS.


RAS antibodies unsuitable for IF of endogenousRAS proteinsWe investigated whether any of the isoform- and mutant-specific RASantibodies validated for use inWestern blotting could also be used forIF detection of RAS proteins in Rasless MEFs (Fig. 5A). Our initial,baseline metric for suitability in IF was the lack of a significant signalin RaslessMEFs expressing BRAFV600E. Only one, the NRAS antibodySC-31, met this criterion (Fig. 5A). Notably, adherent Rasless MEFs ex-pressing NRAS cover large surface areas when cultured on plastic orglass. Upon detaching these cells with trypsin, they are similar in sizeto RaslessMEFs expressing other RAS proteins in suspension.More-over, the NRAS antibody selectively stained RaslessMEFs expressingNRAS but not Rasless MEFs expressing KRAS4A, KRAS4B, or HRAS(Fig. 5B). The subcellular distribution of the signal was consistent withthat of NRAS in our previous reports (32, 33). However, not all cells ineach culture of Rasless MEFs expressing NRAS stained positively, pre-sumably because of differing levels of NRAS expression.

Despite their utility in Western blotting, both RAS mutation–specific antibodies also failed to be suitable for IF (Fig. 5C). The G12D-specific antibody picked up substantial background in Rasless MEFsexpressing BRAFV600E. Although the G12V-specific antibody did notappreciably stain Rasless MEFs expressing BRAFV600E, it did stainRasless MEFs expressing any other RAS isoform. We suspect that theuse of these antibodies in IF may be limited by their low sensitivity, asshown in our Western blots, making it difficult to distinguish endoge-nous levels of RAS from background signals in individual cells.

Table 2. Validated RAS antibodies.

Isoform
Validated antibodies Comments
HRAS/KRAS/NRAS

CPTC–KRAS4B-2
Nonspecific bands above50 kDa, very sensitive
05-1072
Nonspecific bands above 30 kDa
HRAS
18295-1-AP Nonspecific bands above 30 kDa
NRAS
SC-31 May be useful for IF inoverexpression models
KRAS4A/4B
OP24*/SC-30* No IF
KRAS4B
WH0003845M1 Very sensitive, no IF
KRAS4A

ABC1442
Nonspecific bands above 40 kDa,may have difficulty detectingendogenous levels, no IF
SC-522
Nonspecific bands above 30 kDa,may have difficulty detectingendogenous levels, no IF
*Same clone.

Fig. 4. Mutation-specific RAS antibodies detect mutant KRAS proteins inMEFs and human cancer cell lines with high fidelity. Western blotting wasperformed with G12D (top) and G12V (bottom) mutation–specific antibodies to probe(left) Rasless MEFs rescued with the indicated mutants of human KRAS [or wild-type(WT) RAS or BRAF controls] or (right) KRASmutant cancer cell lines. All cell lines exceptHCT116 are homozygous for mutant KRAS. Full (uncut) blots are shown to reveal theabsence of nonspecific protein recognition. Blots were probed with KRAS4B antibodyto show total KRAS protein. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)served as a loading control.

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Similarly, our attempts to detect endogenous wild-type and mutantNRAS in human cancer cell lines by IF, using the same SC-31 antibodythat selectively stained Rasless MEFs expressing NRAS, were un-successful. We first validated byWestern blotting that we could depletethe endogenous NRAS signal in cancer cells harboring wild-typeNRAS (A375 cells) or mutant NRAS (Q61K) (SBC12 cells). Despitenear-complete NRAS knockdown (Fig. 6A), the IF signal remained un-changed in each case (Fig. 6B).

It is widely appreciated that antibodies selected for utility in oneapplication may not perform well in another (27). Western blots areperformed under denaturing conditions, whereas IF detects proteinsin more native conformations. Thus, although disappointing, it maynot be entirely surprising that we have been unable to identify RASantibodies suitable for both Western blotting and IF.

DISCUSSIONThere is now a greater appreciation that there are RAS isoform–distinctbiological roles in normal andneoplastic cells and that there aremutation-distinct functional consequences of these differences for RAS function(9). Research that aims to successfully address these differences requiresthe use of antibody reagents that have been rigorously validated forspecificity and for utility in designated experimental applications. Werecognize that the degree of validation of commercially available RASantibodies is highly variable and that rigorous validation is not feasiblefor most research laboratories to perform on their own. The NCI RASInitiative was established in part to address such gaps in the field (29).


We completed studies that critically as-sessed 22 commercially available RASantibodies, including those most widelyused in the literature. Our goals were toidentify potential limitations of each anti-body, to rigorously validate antibodiesfor use in the field in specific applica-tions, and, ultimately, to establish howaccurately it is possible to interpret dataacquired using each antibody. A criticalcomponent of our analyses was the utili-zation of a unique panel of Rasless MEFsexpressing BRAFV600E or other RAS iso-forms. With this well-validated panel,whichwe established at theNCIRAS Ini-tiative, coupled with our analyses of re-combinant unprocessed and processedhuman KRAS4A/4B proteins and byusing shRNA vectors targeting each RASgene, we were able to very rigorouslyassess RAS specificity and isoform se-lectivity under different experimentalconditions. We have identified anti-bodies that selectively recognize eachof the four human RAS proteins in hu-man cancer cells, thereby identifying aset of RAS antibodies that can be usedwith confidence in future studies usingWestern blot analyses. Our survey didnot identify RAS antibodies useful forother experimental applications suchas IF or IHC.

Consistent with perceptions of the antibody field in general (20–22),we found a broad spectrum of RAS antibody quality, from poor toexcellent. Overall, most of these antibodies displayed their vendor-described specificities, but several vendors provided incomplete or over-stated information that may lead to misinterpretation of data.

Given the high overall amino acid sequence identity of RAS proteins(9), many antibodies generated against one specific RAS isoform willalso recognize other RAS isoforms. For example, although monoclonalantibodyCPTC–KRAS4B-2was generated againstKRAS4B, the nameis misleading in that it suggests KRAS4B selectivity. Instead, we foundthat CPTC–KRAS4B-2 was the most effective pan-RAS antibody avail-able and can provide a useful reagent to determine overall total RASprotein expression in cells. However, this antibody may also recognizeother ~21-kDa small GTPases with sequence similarity to RAS. Becauseectopic expression of other small GTPases cannot restore the growth ofRasless MEFs (31), we do not have reagents to address potential anti-body selectivity for such proteins with similar rigor as done here forRAS. Conversely, one polyclonal antibody generated against recombi-nant NRAS poorly recognized recombinant NRAS, showed strongerrecognition of other recombinant RAS proteins, and failed to detectNRAS in our panel of rescued Rasless MEFs expressing other RASisoforms. Thus, although it did identify a ~21-kDa band in HEK293Tcells, we suspect that the detected band is not NRAS. Finally, althoughthe product information provided with this antibody showed that itrecognizes only a single ~21-kDa band in HEK293 cell lysates, withno recognition of other cellular proteins, our analyses in both RaslessMEFs and HEK293T cells found very significant nonspecific reactivity.

Fig. 5. An IF signal from isoform- or mutation-specific RAS antibodies does not reliably indicate the presenceof the relevant RAS protein. (A) The indicated RAS isoform–selective antibodies were used in IF analyses of RaslessMEFs expressing solely the relevant RAS isoform or BRAFV600E. no 1 Ab, no primary antibody. (B) To determine theRAS isoform selectivity of the NRAS SC-31 signal shown in (A), we used the same antibody to probe Rasless MEFsexpressing each RAS isoform. (C and D) RASG12D or RASG12V mutation-specific RAS antibodies were used for IF analy-ses of Rasless MEFs expressing the relevant KRAS mutant or BRAFV600E negative control (C, KRASG12D) or WT RASisoforms (D, KRASG12V). Scale bars, 20 mm; blue, 4′,6-diamidino-2-phenylindole (DAPI) nuclear stain; green, goat anti-rabbit or anti-mouse Alexa Fluor 488, to detect each primary RAS antibody.

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These and other examples emphasize the critical value of Rasless MEFsin evaluating RAS antibody selectivity and utility in different exper-imental applications.

Of the two KRAS splice variants, KRAS4B has been by far the moststudied in the cancer field, due in large part to availability of reagentsand a widely held perception that the KRAS4B splice variant is thepredominant KRAS protein expressed in human tumor cells (5). How-ever, in the absence of antibodies capable of distinguishing KRAS4Aand KRAS4B, this perception was based on early studies of KRASexpression at the level of mRNA, not protein. Ironically, the very firstdiscovery of KRAS as an oncogene was of the Kirsten murine sarcomavirus, which encodes a G12-mutated rat KRas4A protein (34). Further-more, our studies found that 30 of 30 human cancer cell lines expressboth KRAS4A and KRAS4B mRNA and that, in colorectal carcinomatissues, KRAS4A and KRAS4B transcripts are similarly abundant (6).Our analyses here did identify useful antibodies for selective detectionof KRAS4A and KRAS4B proteins. Regarding KRAS4B-selective anti-bodies, we found that the two commercially available antibodies, al-though clearly selective when evaluated using recombinant proteins,did not effectively recognize cell-expressed KRAS4B protein in RaslessMEFs. One antibody did detect ~21-kDa bands, but the equivalentdetection of such bands in the Rasless MEFs expressing BRAFV600E


demonstrates that this antibody does not detect cellular KRAS4B ex-pression. Instead, we found that the best KRAS4B-selective antibodywas one described as detecting both KRAS4A and KRAS4B. We didvalidate two useful KRAS4A-selective antibodies, with each having dis-tinct nonspecific activities that do not compromise their applicationin Western blots but likely limit their usefulness in other applications.Currently, whether KRAS4A and KRAS4B serve distinct driver roles incancer is not known. To date, the relative expression of the two KRASsplice variants has been determined only at the RNA level. ValidatedKRAS splice variant–selective antibodies will provide crucial reagents toaccurately address this issue. Furthermore, using our validated antibo-dies,we identified shRNAconstructs selective for eachRAS isoform thatcan assist in determining the specificity of additional antibodies.

Finally, we validated two mutation-specific antibodies for Westernblot detection of RAS proteins with G12D or G12Vmutations (whichcomprise 59% of all KRAS mutations), supporting the feasibility ofdeveloping additional antibodies selective for other common RASmutations. Current assays to detect GTP-bound RAS are not amenableto IHCevaluation of patient tumor tissue.Unfortunately, thesemutation-selective antibodies were also not useful for IHC analyses of formalin-fixed, paraffin-embedded or frozen tissue from two cases harboringpreviously identified KRASG12D and KRASG12V mutations, althoughWestern blot analyses of tissue from the same two cases confirmedexpression of the G12D or G12V mutant RAS proteins (fig. S2). Thus,these antibodies cannot selectively measure activated RAS in patienttissue.

In summary, we have performed comprehensive analyses of theselectivity of a large panel of RAS antibodies and achieved our goal ofvalidating antibodies that are useful for Western blot detection of eachof the four RAS proteins as well as of the two KRAS mutations mostprevalent in cancer. Our studies also emphasize the need for well-validated RAS antibodies for other experimental applications, in partic-ular for IF analyses of endogenous RAS proteins, where our currentknowledge of the spatial distribution of RAS proteins has relied exclu-sively on the study of exogenously expressed proteins.With the possibleexception of a validated NRAS-selective antibody, we did not identifyRAS antibodies useful for IF analyses. Although many commerciallyavailable RAS antibodies are described as being useful for IF (Table 1),our findings stress that more rigorous validation of antibody utility inspecific applications, beyond simply detecting signal, needs to be done.We did not evaluate the usefulness of RAS antibodies for IP or (withthe exception of G12mutation–specific antibodies) for IHC, althoughthe inability of the antibodies we tested to perform well in IF suggeststhat they may also not work well in IP applications. We did not eval-uate all commercially available RAS antibodies. Nevertheless, ourWestern blot results can serve as a resource and guide for furtherqualifying RAS antibodies for additional applications.

MATERIALS AND METHODSE. coli expression and purification of human RAS proteinsRecombinant proteins were expressed as His6-MBP (maltose-bindingprotein)–tev (tobacco etch virus)–RAS fusion proteins in E. coli [pRare,BL21 Star (DE3)] and purified, as we described (35). After proteolyticcleavage of the N-terminal tags, the final proteins generated wereGlyGlySerGly-KRAS4B (2 to 188), Gly-HRAS (1 to 189), and Gly-NRAS (2 to 189). Protein concentrations were determined by UV280

(ultraviolet absorbance at 280 nm) using an extinction coefficient of19,865 M−1 cm−1 (11,920 from protein and 7945 from GDP) (36).

Fig. 6. NRAS antibody can detect endogenous NRAS protein by Western blotbut not by IF. (A) Immunoblot showing shRNA knockdown of NRAS protein inA375WT and SBcl2Q61K melanoma cell lines. (B) Representative images of IF analysesperformed as in Fig. 5 but in A375 and SBcl2 cells knocked-down with nonspecific(NS) or NRAS shRNA and co-stained with NRAS (SC-31) antibody and co-localizationmarkers for plasma membrane (wheat germ agglutinin), endoplasmic reticulum[protein disulfide isomerase (PDI)], or Golgi (GM130). Scale bars, 10 mm; blue, DAPInuclear stain; green, RAS; red, organelles.

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Insect cell expression and purification of KRAS4Aand KRAS4BExpression and purification of processed GlyGly-Hs.KRAS4A (2 to189) and GlyGly-Hs.KRAS4B (2 to 188) were performed, as we de-scribed previously, using a bacmid engineered to coexpress farnesyl-transferase subunit a (FNTA) and farnesyltransferase subunit b(FNTB) (35). Expression of nonprocessed GlyGly-Hs.KRAS4A andGlyGly-Hs.KRAS4B followed the same protocol except that the expres-sion constructs were cloned into the standard bacmid. Purification ofprocessed KRAS4B was performed, as we described (35), whereas pro-cessed KRAS4A was purified using a modified protocol that added ananion exchange step after the initial capture from lysate. NonprocessedKRAS4B was purified, as we described previously (30).

Establishment of Rasless MEFs with isoform-specific RASprotein productionA clonal panel of MEF cell lines containing single-transgene alleles wasgenerated using the Rasless MEF system developed by Drosten et al.(31).MEFs null for bothHras andNras (provided byM. Barbacid) weretreated with 600 nM 4-hydroxytamoxifen (4-OHT) for 9 to 11 days toremove the endogenous floxedKRAS gene. Cells lacking all endogenousRas proteins arrest in G1 but resume proliferation upon lentiviral deliv-ery of an ERK (extracellular signal–regulated kinase) MAPK (mitogen-activated protein kinase) pathway activating gene (for example, RAS orBRAF). Alternatively, cell line pools were generated through lentiviraltransduction and antibiotic selection followed by treatment with 4-OHT and single-cell cloning. A single-cell clone was validated for eachtransgene. Confirmation of endogenous Kras gene removal was per-formed by Western blotting and sequencing.

RAS antibody generationPurified unprocessed full-length wild-type KRAS4B (30) was submittedfor antibody generation at Precision Antibody. Candidate clones werescreened at the Antibody Characterization Laboratory (ACL; FNLCR)following procedures outlined on the antibody portal website (http://antibodies.cancer.gov). Four candidates were selected based on reac-tivity by Western blot on a Wes instrument (ProteinSimple) and usingthe IPmass spectrometrymethod developed by the ACL. All antibodiesand characterization data can be found on the ACL web portal, andantibodies and hybridomas are available through theDSHB (Universityof Iowa).

SDS-PAGEEach purified RAS protein was diluted in 1× NuPAGE LDS loadingbuffer (Invitrogen), containing 25 mM tris(2-carboxyethyl)phosphine(TCEP; Sigma-Aldrich) to final protein concentrations of 3, 1, and0.3 mg per 10 ml and applied to 10 to 20% gradient Criterion 26-welltris-HCl precast gels (Bio-Rad) in 1× tris-glycine-SDS running buffer.BenchMark protein ladder size standards (Thermo Fisher Scientific)flank each set of proteins. Gels were stained with SimplyBlue SafeStain(Thermo Fisher Scientific) and then imaged in a Fujifilm LAS-3000equipped with an LAS-4000 CCD camera.

Western blotsFor purified protein, each RAS protein was diluted in 1×NuPAGE LDSloading buffer containing 25 mMTCEP to final protein concentrationsof 10, 1, and 0.1 ng per 10 ml and denatured by heating to 70°C for10 min. Western blots were performed, as described previously (37).Cells were lysed in 1% Triton detergent buffer and assayed for protein


content using the bicinchoninic acid assay (Pierce Biotechnology).Twenty micrograms of each lysate in NuPAGE LDS sample bufferwas loaded into wells of 12% Criterion TGX Stain-Free Gels (Bio-Rad)to the right of aMagicMarkXPprotein ladder (ThermoFisher Scientific).The resolved proteins were transferred to methanol-activated poly-vinylidene difluoride membranes for 90 min in 1× transfer buffer(25mMtris, 94mMglycine, and 20%methanol) at currentsmaintainedbetween 350 and 450 mA. After blocking in 3% nonfat dry milk andwashing in TBS-T (tris-buffered saline with Tween 20), membraneswere incubated overnight in 10 mg of primary antibody diluted in 10mlof 3%drymilk in 1×TBS-T.Membraneswerewashed again and rockedat room temperature for 1 hour in horseradish peroxidase–labeledsecondary antibody (horse anti-mouse, #7076 or goat anti-rabbit,#7074; Cell Signaling Technology) diluted 1:2000 in 3% dry milk inTBS-T. After washing, membranes were developed with AmershamECL Prime Western Blotting Detection Reagent (GE Healthcare) in aChemiDoc Imaging System (Bio-Rad). Anti-vinculin (V9131) or anti-GAPDH (G8795) antibodies (1:5000; Sigma-Aldrich) were used asloading controls. Blots were not stripped before probing for loadingcontrols. A full list of RAS antibodies can be found in Table 1.

Cell cultureRescued Rasless MEFs, generated at FNLCR as described above, weremaintained in Dulbecco’s modified Eagle’s medium (DMEM; CorningInc.) supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich).Established human cancer cell lines were obtained from the AmericanType Culture Collection andmaintained in RPMI 1640 (NSCLC cancerlines, Corning Inc.) or in DMEM (melanoma cell lines, Corning Inc.)supplemented with 10% FBS. Cell line identities were confirmed byshort tandem repeat analysis.

shRNA knockdowns of specific RAS isoformsLentivirus virus-like particles expressing shRNAs targeted againstdifferent RAS transcripts were produced in HEK293T cells by transfec-tion of a 4:3:1 ratio of NRAS (or nonspecific) shRNA plasmid/psPAX2/pMD2.G and a 1:3 ratio of DNA/FuGENE 6 (Promega). Sequencestargeted by each shRNA are provided in table S1.

Immunofluorescent laser scanning confocal microscopyAll rescued Rasless MEFs expressing BRAFV600E or RAS isoforms wereplated at a density of 4 × 104 cells in 12-well no. 1.5 MatTek plates with14-mm coverslips (MatTek Corporation) and grown for 24 to 48 hoursbefore fixing in 2% paraformaldehyde for 15 min at room temperature,4% paraformaldehyde for 5 min at room temperature, or 100% meth-anol for 20 min at −20°C. Paraformaldehyde-fixed cells were permea-bilized with 0.1% Triton X-100 in 1× phosphate-buffered saline for15 min. All cells were then incubated in blocking buffer (Rockland)containing 0.1% Triton X-100 for 45 min at room temperature. Cellswere incubated overnight in 1:100 dilutions of each RAS antibody. Cellswere then incubated in 1:300 goat anti-mouse or goat anti-rabbit AlexaFluor 488 (Invitrogen) in blocking buffer + 0.1% Triton X-100. Tenminutes before completion of secondary antibody incubation, a 1:100dilution of DAPI (50 mg/ml; Thermo Fisher Scientific) was added toeach well to stain nuclei. After washing, cells were imaged in a ZeissLSM700 scanning confocal microscope with a 40× oil immersion lens.Images were analyzed using ZEN blue software (Carl Zeiss AG).Changes in fixation method did not affect localization, except with anti-HRAS. Methanol fixation of Rasless MEFs expressing HRAS led to lossof nuclear staining in both Rasless MEFs expressing BRAFV600E or

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NRAS. Otherwise, the nonspecific signal was preserved. All imagesshown are from cells fixed in 2% paraformaldehyde, except forWH0003845M1 KRAS images, which were from methanol-fixed cells.

For co-localization experiments in melanoma lines, A375 and SBC12cells were fixed in 2%paraformaldehyde for 15min at room temperature.To visualize the plasma membrane, we added wheat germ agglutininAlexa Fluor 647 conjugate (W33466, Thermo Fisher Scientific) labelingsolution (5 mg/ml in 250 ml) to each well and incubated it for 10 min at37°C. For endoplasmic reticulum or Golgi labeling, anti-PDI (MA3-019,Invitrogen) or anti-GM130 (610823) (BD Biosciences) was incubatedon the cells overnight (1:300), followed by goat anti-mouse Alexa Fluor568 for 45min at room temperature. Cells werewashed and imaged in aZeiss LSM700 with a 63× oil immersion lens. Images were analyzedusing ZEN blue software.

IHC on formalin-fixed and frozen sections of humantumor tissuesIHC was performed on 4-mm-thick formalin-fixed, paraffin-embeddedde-identified patient tumor sections using the Discovery XT automatedsystem (VentanaMedical Systems). Sections were mounted on chargedglass slides and baked at 60°C for 1 hour before heat-induced antigenretrieval was performed using standard CC1 (Cell Conditioning 1 so-lution). Slides were incubated with G12D (14429)– or G12V (14412)–selective antibodies for either 2 hours at room temperature (UltraMapprotocol) or 1 hour with heat (DABMap protocol). Chromogenic de-tection was done with the DABMap kit or the UltraMapDAB anti-Rbkit. Frozen sections (4 mm thick) were fixed in neutral buffered formalinfor 10min before the assay was performed on theDiscovery Ultra auto-mated system (Ventana Medical Systems). Slides underwent 36 minof CC1 treatment before incubation with G12D or G12V antibodies,titrated at 1:100, for 60 min at 37°C. Detection was completed withthe DABMap kit.


g/

SUPPLEMENTARY MATERIALSwww.sciencesignaling.org/cgi/content/full/10/498/eaao3332/DC1Fig. S1. RAS isoform siRNA and shRNA binding sites.Fig. S2. IHC analyses using mutation-specific RAS antibodies in tumor tissues harboringKRASG12D or KRASG12V.Table S1. RAS shRNA sequences.Table S2. RAS siRNA sequences.References (38, 39)

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Acknowledgments: We thank P. Ariel and the Microscopy Services Laboratory at University ofNorth Carolina at Chapel Hill and B. Papke and K. Bryant from the Der Lab for technicalassistance with confocal microscopy. We acknowledge the following personnel from the NCIRAS Initiative at the FNLCR for their technical expertise in cloning, expression, purification, andquality control of materials: V. Wall, J. Mehalko, C. Grose, C. Bittner, T. Taylor, M. Sherekar,S. Messing, P. Frank, S. Perkins, R. Gapud, and Z. Meng. We thank D. G. Wong and A. N. Karnezisfor assistance with the IHC analyses. Funding: This work was supported by grants (CA42978,CA179193, CA175747, and CA199235 to C.J.D. and A.D.C., CA197709 to F.M., and CA165995to P.J.H.) and training grants/fellowships (T32 CA009156 to A.V.V. and G.A.H., F32 CA200313 toG.A.H., and a grant from the American Cancer Society to A.V.V.) from the NIH/NCI and thePancreatic Cancer Action Network–American Association for Cancer Research. C.J.D. was alsosupported by a grant from the Department of Defense and from the Lustgarten Pancreatic


Cancer Foundation. A.M.W., W.K.G., L.A.S., N.F., G.R.W., R.B., F.M., and J.L.H. were funded in wholeor in part with federal funds from the NIC/NIH under contract no. HHSN261200800001E. Thecontent of this publication does not necessarily reflect the views or policies of the Departmentof Health and Human Services, nor does mention of trade names, commercial products, ororganizations imply endorsement by the U.S. Government. Author contributions: A.M.W.,A.D.C., C.J.D., J.L.H., A.V.V., G.A.H., B.T.-C., and D.G.H. designed the experiments. A.M.W., A.D.C.,C.J.D., and J.L.H. wrote the manuscript. A.M.W., I.O.-D., A.V.V., N.F., and B.T.-C. performedthe experiments. W.K.G., N.F, L.A.S., R.B., P.J.H., and G.R.W. generated tools, cell lines, andreagents for the experiments. M.R.P. and F.M. provided scientific guidance. Competinginterests: The authors declare that they have no competing interests. Data and materialsavailability: Rasless MEFs expressing BRAFV600E or RAS proteins are available through anMTA from the FNLCR. Antibodies generated by the ACL at the FNLCR are available from theDSHB at the University of Iowa.

Submitted 9 July 2017Accepted 6 September 2017Published 26 September 201710.1126/scisignal.aao3332

Citation: A. M. Waters, I. Ozkan-Dagliyan, A. V. Vaseva, N. Fer, L. A. Strathern, G. A. Hobbs,B. Tessier-Cloutier, W. K. Gillette, R. Bagni, G. R. Whiteley, J. L. Hartley, F. McCormick,A. D. Cox, P. J. Houghton, D. G. Huntsman, M. R. Philips, C. J. Der, Evaluation of theselectivity and sensitivity of isoform- and mutation-specific RAS antibodies. Sci. Signal. 10,eaao3332 (2017).

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Evaluation of the selectivity and sensitivity of isoform- and mutation-specific RAS antibodies

Peter J. Houghton, David G. Huntsman, Mark R. Philips and Channing J. DerTessier-Cloutier, William K. Gillette, Rachel Bagni, Gordon R. Whiteley, James L. Hartley, Frank McCormick, Adrienne D. Cox, Andrew M. Waters, Irem Ozkan-Dagliyan, Angelina V. Vaseva, Nicole Fer, Leslie A. Strathern, G. Aaron Hobbs, Basile

DOI: 10.1126/scisignal.aao3332 (498), eaao3332.10Sci. Signal.

Their findings show how reliably one can interpret the data acquired from each reagent.for their utility to detect the distinct RAS isoforms in various cell types and for their use in specific analytical methods.

. rigorously assessed 22 commercially available RAS antibodieset alantibodies against RAS is highly variable. Waters always reliable or universally valid for the methods in which they are used; in particular, the reliability of commercialinformation that provides critical insight into both normal and pathological cellular functions. However, antibodies are not

Researchers rely largely on antibodies to measure the abundance, activity, and localization of a protein,A resource on RAS antibodies

ARTICLE TOOLS http://stke.sciencemag.org/content/10/498/eaao3332

MATERIALSSUPPLEMENTARY http://stke.sciencemag.org/content/suppl/2017/09/22/10.498.eaao3332.DC1

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REFERENCES

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Evaluation of the selectivity and sensitivity of isoform ... · Evaluation of the selectivity and sensitivity of isoform- and mutation-specific RAS antibodies Andrew M. Waters,1,2