Antibodies, Kits, and Reagents for Apoptosis Research · Staurosporine #9953: WB analysis of extracts from HeLa cells, untreated or Staurosporine-treated (3 hr), showing PARP cleavage

CELL SIGNALING TECHNOLOGY

Antibodies, Kits, and Reagents for

Apoptosis Research

Apoptosis is a tightly controlled pattern of cell death characterized by nuclear condensation, cell shrinkage, membrane blebbing, and DNA fragmentation. The central regulators of apoptosis are the caspases, a family of cysteine proteases that fall into two broad categories. The “initiator” caspases-2, -8, -9, -10, and -12 are closely coupled to upstream, pro-apoptotic signals and act by cleaving and activating the downstream “executioner” caspases-3, -6, and -7 that modify proteins ultimately responsible for programmed cell death. Targets such as PARP and lamin A/C are cleaved by executioner cas-pases and serve as markers of apoptosis.

There are two primary apoptotic pathways that lead to caspase activation: the intrinsic and extrinsic path-ways. The intrinsic pathway is activated by cell stress, DNA damage, developmental cues, and withdrawal of survival factors. This pathway is regulated by the Bcl-2 family of proteins, which consists of pro-apoptotic (Bax, Bak, Bad, Bid, Puma, Bim, and Noxa) and anti-apoptotic (Bcl-2, Bcl-xL, Bcl-w, Mel-1) factors. Bcl-2 family mem-bers localize to the mitochondria where they control

mitochondrial permeability, release of cytochrome c, and activation of initiator caspase-9 followed by execu-tioner caspase-3. Intrinsic apoptosis can be inhibited through signaling pathways that promote survival, such as the PI3K/Akt and MAPK pathways.

Caspases can also be activated through the extrinsic or death receptor pathway. The death receptors con-sist of members of the TNFR family (TNFR1/2, Fas, and DR3/4/5) and their associated ligands (TNF-α, FasL, TRAIL, TWEAK). Ligand binding induces receptor acti-vation, leading to signaling through the adaptor proteins FADD and TRADD to activate the initiator caspase-8 and the executioner caspase-3. The death receptor pathway can also lead to cell survival through TNFR2-mediated signaling to NF-κB, which induces expression of pro-survival genes, Bcl-2 and FLIP.

Select Reviews Dickens, LS, Powley, IR, Hughes, MA, et al. (2012) Exp. Cell Res. 318, 1269-1277. | Favaloro, B, Allocati, N, Graziano, V, et al. (2012) Aging 4, 330-349. | McIlwain, DR, Berger, T, and Mak, TW. (2013) Cold Spring Harb. Perspect. Biol. 5, a008656. | Shamas-Din, A, Kale, J, Leber B, et al. (2013) Cold Spring Harb. Perspect. Biol. 5, a008714.

Apoptosis

INTRODUCTION

www.cellsignal.com/apoptosis

A Trusted Research PartnerCell Signaling Technology (CST) strives to be your research partner for the study of apoptosis. As scientists we understand the importance of using antibodies that consistently work each and every time. Our highly specific antibodies are directed against the most relevant targets in apoptosis and are painstakingly validated to work in their recommended applications, so you can feel confident in your results. In addition, we provide siRNA, chemical modulators, control cell extracts, and kits—all validated using the same rigorous quality standards—giving you the tools you need for every step of the experimental process. Optimal antibody dilutions and recommended buffers are predetermined for you, saving you the time, sample, and trouble of additional optimization steps. Protocols and troubleshooting guides for commonly used applications are available on our website to help you get reliable results in the shortest amount of time. If you experience a problem in the lab, the same expert scientist who produced and validated your antibody will respond to your email or phone call and help you, sharing their bench experience and data from their notebooks.

Shuji, Scientist, has been with CST since 2008.

4 Tools and Targets

6 Intrinsic Pathway Tools

8 Extrinsic Pathway Tools

10 Apoptosis and Cancer

14 Validation and Support

Table of Contents

4

TOOLS AND TARGETS

Products and Toolsfor Apoptosis ResearchCST offers the highest possible quality antibodies and reagents for each stage of the experimental process.

A1/Bfl-1

Acinus

Phospho-Ack1 (Tyr284)

AIF

Alix

AP-2α, -2β, -2γPhospho-AP2M1 (Thr156)

Apaf-1

APR3

Aven

Bad

Phospho-Bad (Ser112), (Ser136),

(Ser155)

Bak

BAP31

Bax

Bcl-2

Phospho-Bcl-2 (Thr56), (Ser70)

Bcl-w

Bcl-xL

BCL2L10

BID

Bik

Bim

Phospho-Bim (Ser55), (Ser69),

(Ser77)

BIRC6

Bmf

BNIP3

BNIP3L

Phospho-BNIP3L (Ser62)

Bok

c-IAP1

c-IAP2

Caspase-1

Cleaved Caspase-1 (Asp297)

Caspase-2

Caspase-3


Caspase-4

Caspase-5

Caspase-6


Caspase-7


Caspase-8

Cleaved Caspase-8 (Asp374),

(Asp384), (Asp391)

Caspase-9

Phospho-Caspase-9 (Thr125), (Asp315),

(Asp330), (Asp353)

Caspase-12

Caspase-14

Caspase Cleavage Motif

Cleaved Caspase Substrate

CRADD/RAIDD

Cytochrome c

Acetyl-Cytochrome c (Lys8)

DAP1

DAP3

DAPK1

DAPK3/ZIPK

Daxx

Cleaved Drosophila Dcp-1 (Asp216)

DcR1

DcR2

DcR3

DFF45/DFF35

Cleaved DFF45 (Asp224)

DIDO1

DR3

DR4

DR5

Phospho-DR6 (Ser562)

DRAK2

We’ve got it coveredOur total and modification-specific antibody collection provides comprehensive coverage of the apoptotsis pathways.

Primary Antibodies CST offers a broad range of highly specific primary antibodies to key targets in apoptosis signaling pathways. Currently, we offer more than 300 primary antibodies against over 150 protein targets, including phosphorylation site and cleavage-specific antibodies. Our portfolio is constantly expanding, so please check our website frequently for a complete, up-to-date product list.

Antibody Sampler Kits Antibody Sampler Kits allow for comprehensive coverage of multiple nodes in a pathway of interest.

Assay Kits Assess apoptosis using a CST assay kit, scale up your analysis in a 96-well format using PathScan® ELISA Kits (384-well plates are also available on a custom basis), or monitor multiple pathway nodes in parallel using sandwich assays in a slide-based array.

siRNA SignalSilence® siRNA allows you to effectively knockdown proteins of interest.

Chemical Modulators Treat cells with a chemical modulator to induce apoptosis and examine effects on downstream signaling.

Experimental Controls

Control cell extracts, control proteins, blocking peptides, and isotype controls can help verify antibody specificity, critical for accurate data analysis.

Companion Products Whatever your intended application, CST has a wide selection of companion products to support your experiment such as secondary antibodies, loading controls, buffers, dyes, or other detection reagents. Our companion products work optimally with our antibodies and save time by having all the tools you need in one place.


Apoptotic Stimuli

Treat cells with a chemical modulator

to induce apoptosis

kDa200140

100

80

60

50

Cleaved PARP

0 1 0.3 0.1 Staurosporine (µM)

200140

100

80

60

50

Full Length PARP Cleaved PARP

Staurosporine #9953: WB analysis of extracts from HeLa cells, untreated or Staurosporine-treated (3 hr), showing PARP cleavage as evidence of induction of apoptosis, using Cleaved PARP Antibody #9541 (upper) or PARP Antibody #9542 (lower).

Experimental Controls

Control cell extracts and siRNAs to confirm

target specificity

kDa

α-Tubulin

200

140

100

80

6050

40

30

20

6050

Caspase-3 siRNA– + +

Caspase-3

III

SignalSilence® Caspase-3 siRNA I (Mouse Specific) #6488 and Signal-Silence® Caspase-3 siRNA II (Mouse Specific) #6501: WB analysis of extracts from NIH/3T3 cells, transfected with 100 nM SignalSilence® Control siRNA (Unconjugated) #6568 (-), #6488 (+), or #6501 (+), using Caspase-3 (8G10) Rabbit mAb #9665 (up-per) or α-Tubulin (11H10) Rabbit mAb #2125 (lower). The caspase-3 (8G10) Rabbit mAb confirms silencing of Caspase-3 expression, while the α-Tubulin (11H10) Rabbit mAb is used as a loading control.

Assays Kits

Apoptosis Detection, ELISA, and Multi-Target Array Kits for experimental analysis

kDa

Cleaved PARP(ASP24)

STO-treated

200140

100

80

6050

40

Untreated

mg/mL1.33

.665

.333

.166

.083

.042

.021

1.33

.665

.333

.166

.083

.042

.021

STO-treatedUntreated

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

0.00 0.25 0.50 0.75 1.00 1.25

Abso

rban

ce45

0 nm

Protein conc. of lysate (mg/ml)

kDa

Cleaved PARP(ASP24)

STO-treated

200140

100

80

6050

40

Untreated

mg/mL1.33

.665

.333

.166

.083

.042

.021

1.33

.665

.333

.166

.083

.042

.021

STO-treatedUntreated

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

0.00 0.25 0.50 0.75 1.00 1.25

Abso

rban

ce45

0 nm

Protein conc. of lysate (mg/ml)

PathScan® Cleaved PARP (Asp214) Sandwich ELISA Kit #7262: The relationship between protein concentration of lysates from untreated and staurosporine (STO) treated HeLa cells and the absorbance at 450 nm is shown. HeLa cells (80% confluent) were treated with staurosporine (1 μM) for 3 hours. Absorbance at 450 nm is shown in the top figure, while the corresponding WB using Cleaved PARP Antibody #9548 is shown in the bottom figure.

Tools to Support Your Apoptosis Workflow

eIF4G2/p97

Endonuclease G

FADD

Phospho-FADD (Ser191),

(Ser194)

FAF1

FAIM

Fas

FasL

FLIP

α-Fodrin

Cleaved α-Fodrin (Asp1185)

Granzyme A

Granzyme B

HIPK2

HtrA2/Omi

Drosophila ICE

Cleaved Drosophila ICE (Asp230)

Lamin A/C

Phospho-Lamin A/C (Ser22)

Cleaved Lamin A (Asp230),

(Small Subunit)

Lamin B1

Lamin B2

LAP2α

Livin

Mad-1

Maspin

Max

Mcl-1

Phospho-Mcl-1 (Ser64),

(Ser159/Thr163)

Mst1

Mst2

Phospho-Mst1 (Thr183)/Mst2 (Thr180)

Mst3

Mst3b

Mst4

c-Myc

Phospho-c-Myc (Thr58/Ser62)

N-Myc

PAR-4

Phospho-PAR-4 (Thr163)

PARP

Cleaved PARP (Asp214)

PDCD4

PEA-15

Phospho-PEA-15 (Ser104)

Perforin

PHLDA3

Puma

Siva-1

Smac/Diablo

Survivin

Phospho-Survivin (Thr34)

TANK

TAX1BP1

Thymidine Phosphorylase/ECGF1

TMS1

TNF-R2

TP/ECGF1

TRADD

TRAF1

TRAF2

TRAF3

TRAF6

TRAIL

VDAC1

VDAC2

WWOX

XAF1

XIAP

Cellular Readouts

Antibodies to key targets as cellular readouts

for apoptosis

Cleaved Caspase-3 (Asp175) (D3E9) Rabbit mAb (Alexa Fluor® 647 Conjugate) #9602: Confocal IF analysis of HeLa cells, untreated (upper) or treated with Staurosporine #9953 (1 μM, 4 hr; lower), us-ing #9602 (blue pseudocolor). Actin filaments were labeled with Alexa Fluor® 488 Phalloidin #8878 (green). Red = Propidium Iodide (PI)/RNase Staining Solution #4087.

6

Casp-9

Casp-3

Bak

tBID

tBID

Bim

Bmt

Bmt

BID

Bax

BaxBak

Bax

Bax

Bad

Bcl-2

Bcl-2

Bcl-2

Bcl-2Puma

Bcl-xL

Bcl-xL

PKC

Erk1/2

p90RSK p70 S6KAktPKA

14-3-3Bad

14-3-3

BadApaf-1

Cyto c

Arts

XIAP Smac/Diablo

AIFATM/ATR

JNK

JNK

p53

PI3K

Bim

Casp-8,-10

FADD

ub

Bak

Mcl-1

HECTH9

HECTH9

PIDD

RAIDD

CaMKII

HrkDP5

HrkDP5

DLC2

LC8

HSP60 HtrA2

Endo G

ING2

SirT2

ARD1NatA

Fas/CD95

FasL

Noxa

Mcl-1

Calcineurin

[NAD]

Casp-2

Microtubules

Acetyl-CoA

ApoptosisDNA Damage

Genotoxic Stress

CytosolicSequestration

Survival Factors:Growth Factors, Cytokines, etc.

Death Stimuli:Survival Factor Withdrawal

INTRINSIC PATHWAY TOOLS

Intrinsic Apoptosis:Bcl-2 ProteinsBroad range of tools to help you examine apoptosis from beginning to end

The Bcl-2 family of proteins regulates apoptosis by controlling mitochondrial permeability. The anti-apoptotic proteins Bcl-2 and Bcl-xL reside in the outer mitochondrial wall and inhibit cytochrome c release. The pro-apoptotic Bcl-2 proteins Bad, BID, Bax, and Bim may reside in the cytosol but translocate to the mitochondria following death signaling, where they promote the release of cytochrome c. Bad translocates to mitochondria and forms a pro-apoptotic complex with Bcl-xL. This translocation is inhibited by survival factors that induce the phosphorylation of Bad, leading to its cytosolic sequestration. Cytosolic BID is cleaved by caspase-8 following signaling through Fas; its active fragment (tBID) translocates to mitochon-dria. Bax and Bim translocate to mitochondria in response to death stimuli, including survival factor withdrawal. Activated following DNA damage, p53 induces the transcription of Bax, Noxa, and Puma. Upon release from mitochondria, cytochrome c binds to Apaf-1 and forms an activation complex with caspase-9. Although the mechanism(s) regulating mitochondrial permeability and the release of cytochrome c during apoptosis are not fully understood, Bcl-xL, Bcl-2, and Bax may influence the voltage-dependent anion channel (VDAC), which may play a role in regulating cytochrome c release. HECTH9 is a DNA damage-activated E3 ubiquitin ligase for p53, and Mcl-1 is an anti-apoptotic member of the Bcl-2 family.

Select Reviews Brenner, D and Mak, TW. (2009) Curr. Opin. Cell Biol. 21, 871–877. | Chalah, A and Khosravi-Far, R. (2008) Adv. Exp. Med. Biol. 615, 25–45. | Lindsay, J, Esposti, MD, and Gilmore, AP. (2011) Biochim. Biophys. Acta.

1813, 532–539. | Ola, MS, Nawaz, M, and Ahsan, H. (2011) Mol. Cell. Biochem. 351, 41–58. | Pradelli, LA, Bénéteau, M, and Ricci, JE. (2010) Cell. Mol. Life Sci. 67, 1589–1597. | Rong, Y and Distelhorst, CW.

(2008) Annu. Rev. Physiol. 70, 73–91. | Speidel, D. (2010) Trends Cell Biol. 20, 14–24. | Suen, DF, Norris, KL, and Youle, RJ. (2008) Genes Dev. 22, 1577–1590.


Antibodies to key targets as cellular readouts

Bak (D4E4) Rabbit mAb #12105: Confocal IF analysis of OVCAR8 cells showing colocalization with mitochondria using #12105 (green; A) and MitoTracker® Red CMXRos (Red; B). Merged image (C). Blue pseudocolor = DRAQ® #4084 (fluorescent DNA dye).

A B C


kDa

46.5

28

20.5

5776

14.5

Cleaved Caspase-3

– + Cytochrome c

6.5

46.5

28

20.5

5776

14.5

6.5

Pro-Caspase-3

#9662

#9661

Experimental controls for accurate data analysis

Caspase-3 Control Cell Extracts #9663: WB analysis of Jurkat cell extracts untreated or treated with cytochrome c in vitro, showing full length and/or cleaved caspase-3 (upper) and cleaved caspase-3 Asp175 (lower), us-ing Caspase-3 Antibody #9662 and Cleaved Caspase-3 (Asp175) Antibody #9661.

Pro-Apoptosis Bcl-2 Family Antibody Sampler Kit #9942

Antibody sampler kits offer a convenient and economical means for the analysis of multiple nodes within a pathway of interest.

Kit Includes: Bad (D24A9) Rabbit mAb #9239, Phospho-Bad (Ser112) (40A9) Rabbit mAb #5284, Bax (D2E11) Rabbit mAb #5023, Bik Antibody #4592, Bim (C34C5) Rabbit mAb #2933, BID Antibody (Human Specific) #2002, Bak (D4E4) Rabbit mAb #12105, Puma (D30C10) Antibody #12450, Anti-rabbit IgG, HRP-linked Antibody #7074

For a complete listing of our Antibody Sampler Kits:

www.cellsignal.com/abkits

SignalSilence® siRNA for knockdown studies kDa

Bcl-xL

α-Tubulin

140

100

80

6050

6050

40

30

20

III

Bcl-xL siRNA– + +

SignalSilence® Bcl-xL siRNA I #6362: WB analysis of extracts from HeLa cells, transfected with 100 nM SignalSilence® Control siRNA (Unconjugated) #6568 (-), #6362 (+), or SignalSilence® Bcl-xL siRNA II #6363 (+), using Bcl-xL (54H6) Rabbit mAb #2764 (upper) or α-Tubulin (11H10) Rabbit mAb #2125 (lower). The Bcl-xL (54H6) Rabbit mAb confirms silencing of Bcl-xL expression, while the α-Tubulin (11H10) Rabbit mAb is used as a loading control.

kDa

Cleaved PARP(Asp214)

PARPCleaved PARP(Asp214)

140

100

8060

5040

140

100

80

60

5040

Etoposide– +

Chemical modulators to induce apoptosis

Etoposide #2200: WB analysis of extracts from Jurkat cells, untreated (-) or Etoposide-treated (25 μM, overnight; +), using Cleaved PARP (Asp214) (D64E10) XP® Rabbit mAb #5625 (upper) or total PARP Antibody #9542 (lower).

Bcl-2

Phospho-Bcl-2(Ser70)

Total Bcl-20

Paclitaxel-treated

1

2

3

4

Abso

rban

ce45

0nm Untreated

λ phosphatase-treated

Paclitaxel– – +


Paclitaxel– – +λ Phosphatase– –+λ Phosphatase– –+

Bcl-2


Total Bcl-20

Paclitaxel-treated

1

2

3

4

Abso

rban

ce45

0nm Untreated


Paclitaxel– – +


Paclitaxel– – +λ Phosphatase– –+λ Phosphatase– –+

PathScan® Phospho-Bcl-2 (Ser70) Sandwich ELISA Kit #11874: Treatment of Jurkat cells with Paclitaxel stimulates phosphorylation of Bcl-2 at Ser70, as detected by #11874, but does not affect the levels of total Bcl-2 detected by PathScan® Total Bcl-2 Sandwich ELISA Kit #12030. Jurkat cells were untreated or treated with λ phosphatase or Paclitaxel #9807 (1 mM, 20 hr, 37°C). The absorbance readings at 450 nm are shown in the left figure, while the corresponding WBs (right) using Phospho-Bcl-2 (Ser70) (5H2) Rabbit mAb #2827 (upper) or Bcl-2 (D55G8) Rabbit mAb (Human Specific) #4223 (lower) are shown.

PathScan® ELISA kits for experimental analysis Bcl-2


Total Bcl-20

Paclitaxel-treated

1

2

3

4

Abso

rban

ce45

0nm Untreated


Paclitaxel– – +


Paclitaxel– – +λ Phosphatase– –+λ Phosphatase– –+ λ phosphatase+ - -

λ phosphatase+ - -

Cleaved Caspase-3 (Asp175) (D3E9) Rabbit mAb (PE Conjugate) #12768: Flow cytometric analysis of Jurkat cells, untreated (blue) or treated with Etoposide #2200 (green), using #12768 (fluorescent DNA dye).

Even

ts

Cleaved Caspase-3 (Asp175) (PE Conjugate)

8

GAS2 ROCK1 Acinus

DFF40

TNF-R1 TNF-R2 DR3APO-3

DR4/5Fas/CD95 TRADD

FADD

CYLDRIPTRADD TRADD

RIP

FasL

APO-3L/TWEAK APO-2L/

TRAIL

RelB

RelB

p65/RelA

p52

IKKα/β/γ

IKKαRIP1

Apaf1

XIAP XIAP

MKK1

JNKRIP3

RIP1

RIP1

Casp-8, -10

Casp-8,-10

ASK1

Lamin A Actin α-Fodrin DFF45 PARP

Casp-7Casp-6

Casp-9

Casp-8

Casp-3

FADD

FLIP

Cyto c

TRAF2

TRAF3

cIAP1/2

cIAP1/2

cIAP1/2FADD

TRA

F2TR

AF1

TRAF2 TRAF1

FADD

Daxx

ComplexIIa

ComplexIIb

TNF-α TNF-α

Necroptosis

NIK

IκB

IκBSmac

NF-κB

NF-κBp100

NF-κBp50

MLKL

Bcl-2

Bcl-2

tBID

BID

Cell ShrinkageMembrane Blebbing DNA

FragmentationChromatin

Condensation Transcription

Processing

Apoptosis Survival

Death Receptor Signaling

www.cellsignal.com

© 2002 – 2013 Cell Signaling Technology, Inc. Death Receptor Signaling • created January 2002 • revised February 2012

EXTRINSIC PATHWAY TOOLS

Apoptosis can be induced through the activation of death receptors including Fas, TNFαR, DR3, DR4, and DR5 by their respective ligands. Death receptor ligands characteristically initiate signaling via receptor oligomerization, which in turn results in the recruitment of specialized adaptor proteins and activation of caspase cascades. Binding of FasL induces Fas trimerization, which recruits initiator caspase-8 via the adaptor protein FADD. Caspase-8 then oligomerizes and is activated via autocatalysis. Activated caspase-8 stimulates apoptosis via two parallel cascades: it can directly cleave and activate caspase-3, or alternatively, it can cleave Bid, a pro-apoptotic Bcl-2 family protein. Truncated BID (tBID) translocates to mitochondria, inducing cytochrome c release, which sequentially activates caspase-9 and -3. TNF-α and DR-3L can deliver pro- or anti-apoptotic signals. TNFαR and DR3 promote apoptosis via the adaptor proteins TRADD/FADD and the activation of caspase-8. Interaction of TNF-α with TNFαR may activate the NF-κB pathway via NIK/IKK. The activation of NF-κB induces the expression of pro-survival genes including Bcl-2 and FLIP, the latter can directly inhibit the activation of caspase-8. FasL and TNF-α may also activate JNK via ASK1/MKK7. Activation of JNK may lead to the inhibition of Bcl-2 by phosphorylation. In the absence of caspase activation, stimulation of death receptors can lead to the activation of an alternative programmed cell death pathway termed necroptosis.

Declercq, W, Vanden Berghe, T, and Vandenabeele, P. (2009) Cell 138, 229–232. | Fuchs, Y and Steller H. (2011) Cell 147, 742–758. | Kantari, C and Walczak, H. (2011) Biochim. Biophys. Acta. 1813, 558–563. | Kaufmann, T, Strasser, A, and Jost, PJ. (2012) Cell Death Differ. 19, 42–50. | Lavrik, IN and Krammer, PH. (2012) Cell Death Differ. 19, 36–41. | Van Herreweghe, F, Festjens, N, Declercq, W, and Vandenabeele, P. (2010) Cell. Mol. Life Sci. 67, 1567–79. | Wajant, H and Scheurich, P. (2011) FEBS J. 278, 862–876.

Extrinsic Apoptosis:Death ReceptorsAntibodies and reagents to support every step of your protocol

www.cellsignal.com/apoptosisPr

opid

ium

Iodi

de

Annexin V-FITC

Live Cells

Late Apoptotic/Necrotic

Cells

Early Apoptotic Cells

Prop

idiu

m Io

dide

Annexin V-FITC

Live Cells


Cells


Untreated Camptothecin-treated

Prop

idiu

m Io

dide

Annexin V-FITC

Live Cells


Cells


Prop

idiu

m Io

dide

Annexin V-FITC

Live Cells


Cells


Untreated Camptothecin-treated

HeLa + StaurosporineHeLa



Cleaved Caspase-3 (Asp175)Cleaved Caspase-7 (Asp198)

PathScan® Stress and Apoptosis Signaling Antibody Array Kit (Chemiluminescent Readout) #12856: HeLa cells were grown to 90% confluency and then either untreated (left panel) or treated with Human Tumor Necrosis Factor-α (hTNF-α) #8902 (100 ng/ml, 20 min; right panel). Cell extracts were prepared and analyzed using #12856. Images were acquired by briefly exposing the slide to standard chemiluminescent film.

PathScan® Array kits for multiplex analysis

DRAQ7™ #7406: FC analysis of unfixed Jurkat cells treated with Staurosporine #9953. Gated population represents DRAQ7™-positive apoptotic cells.

Companion products to support your work

Assay kits to measure key cellular readouts

Annexin V-FITC Early Apoptosis Detection Kit #6592: Confocal IF analysis of live Jurkat cells, untreated (left) or camptothecin-treated (right), using Annexin V-FITC Conjugate (green). Red = Propidium Iodide (fluorescent DNA dye). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

kDa

β-Actin

TNF-R1 siRNA(Mouse Specific)

TNF-R1

200

140

100

80

6050

40

50

40

30

20

10

+– +

I IISignalSilence® TNF-R1 siRNA I (Mouse Specific) #13369: WB analysis of extracts from L929 cells, transfected with 100 nM SignalSilence® Control siRNA (Unconjugated) #6568 (-), SignalSilence® TNF-R1 siRNA I (Mouse Specific) (+) or SignalSilence® TNF-R1 siRNA II (Mouse Specific) #13570 (+), using TNF-R1 (D3I7K) Rabbit mAb (Rodent Specific) #13377 (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower). The TNF-R1 (D3I7K) Rabbit mAb (Rodent Specific) confirms silencing of TNF-R1 expression, while the β-Actin (D6A8) Rabbit mAb is used as a loading control.

SignalSilence® siRNA for knockdown studies

kDa


Cleaved PARP

140200

10080

60

50

40

30

20

140200

10080

60

50

40

30

20hHis6FASL (ng/ml)10 10

00 0.1 1

Full Length PARP

Chemical modulators to induce apoptosis

Human His6Fas Ligand/TNFSF6 (hHis6FasL) #5452: Western blot analysis of extracts from Jurkat cells, untreated or treated with hHis6FasL for 3 hr, using Cleaved PARP (Asp214) Antibody (Human Specific) #9541 (upper) or PARP Antibody #9542 (lower).



Death Receptor Antibody Sampler Kit #8356

Explore our antibody sampler kits—convenient and economical ways to assay several nodes in a common signaling pathway. Visit our website to find over 15 sampler kits available for apoptosis research.

Kit Includes: Fas (C18C12) Rabbit mAb #4233, TNF-R1 (C25C1) Rabbit mAb #3736, TNF-R2 Antibody #3727, DR3 Antibody #3254, DR5 (D4E9) XP® Rabbit mAb #8074, FADD Antibody (Human Specific) #2782, TRADD (7G8) Rabbit mAb #3684, DcR3 Antibody #4758, RIP (D94C12) XP® Rabbit mAb #3493, Anti-rabbit IgG, HRP-linked Antibody #7074

Flow cytometric analysis of Jurkat cells untreated (left) or treated with camptothecin (10uM, 4 hr; right) using #6592.

10

Casp-8,-10

Casp-12

Casp-9

Casp-2

Casp-3,-6,-7 Casp-9

NIKRIP

RIP

TRAF2

TAK1ASK1

BID

NF-κB

NF-κB

NF-κB

IκBα

IκBα

IKKγ

IKKβIKKα

FoxO1

FoxO1

p53

p53

p53

p53 c-Jun

HtrA2

SirT2

XIAP

Arts

14-3-3

APP

DFF40

DFF45

Smac/Diablo

Cyto c

Apaf-1

AIF

HECTH9Mcl-1

Endo G

AIF Endo G

Bcl-2

Bcl-xL

tBID

BaxBak

Bad

p90RSK

cdc2

ROCK ATM/ATR

Chk1/2

JNK

TNF, FasL, TRAILTNF, FasL, TRAIL

PIDDRAIDD

Puma

NoxaBim

PKC

Erk1/2

PI3K

Akt

JNK

JNK

FADD

TRAD

DRI

P1 RIP

FAD

D

TRA

DD

c-IA

P

c-IA

PTR

AF2

TRA

F2TRADD

FADD

Itch

CYLD FLIP

Calpain

DFF40

ER Stress

Trophic Factors

DNA Fragmentation DNA Damage

• Cell shrinking• Membrane blebbing

Cytoplasm

Cytoplasm

Nucleus

Cell Cycle

Cellular Stress

[Ca2+]

FLIPXIAP

BimPumaNoxa

© 2002 – 2013 Cell Signaling Technology, Inc. Overview: Regulation of Apoptosis • created January 2002 • revised November 2011

Apoptosis Overview

www.cellsignal.com

APOPTOSIS AND CANCER

Resisting apoptosis is the one of the six hallmark traits of cancer. Cancer cells have developed unique strategies for cell survival that involve both inhibition of pro-apoptotic factors combined with activation of anti-apoptotic factors. One of the primary pathways involved in cell survival is PI3K/Akt. Akt phosphorylates and inhibits several pro-apoptotic factors such as Bim, Bax, Bad, and FoxO1. Akt also activates the inhibitor of apoptosis protein XIAP and blocks p53-mediated apoptosis through stabilization of MDM2. Mutations that result in constitutive signaling through this path-way provide distinct proliferative and survival advantages for a cancer cell. For example, activating mutations in PI3K3C (a subunit of PI3K), and loss-of-function mutations in PTEN both upregulate Akt activity and are some of the most commonly mutated genes in cancer.

Growth factor signaling through MAPK Erk1/2 can inhibit apoptosis through phosphorylation of Bim at multiple sites, including Ser55, Ser65, and Ser73, which promotes its proteasomal degradation. In addition, signaling through Erk1/2 and PKC to p90RSK leads to phosphorylation and inhibition of Bad and increased expression of the anti-apoptotic factors Bcl-2 and Bcl-xL via CREB. Apoptosis can also be prevented through TNFR-mediated signaling to NF-κB, resulting in induction of XIAP and FLIP.

Select Reviews Delbridge, AR, Valente, LJ, and Strasser, A. (2012) Cold Spring Harb. Perspect. Biol. 4 (11). | Brumatti, G, Salmanidis, M, and Ekert, PG. (2010) Cell. Mol. Life Sci. 67, 1619–30. | Fuchs, Y and Steller, H. (2011) Cell 147, 742–758. | Shortt, J and Johnstone, RW. (2012) Cold Spring Harb. Perspect. Biol. 4 (12) | Hanahan, D and Weinberg, RA. (2011) Cell 144, 646-74.

Survival Signalingin CancerComprehensive pathway coverage to simplify your experimental design


kDa

Phospho-Bim(Ser77)

TPA

BimL

BimS

200140100

80

6050

40

30

20

140

100

80

6050

40

30

10

20

– +–

MCF7Raji

+

BimEL

Phospho-Bim (Ser77) (D4H12) Rabbit mAb #12433: WB analysis of extracts from Raji and MCF7 cells, untreated (-) or treated with TPA #4174 (200 nM, 30 min; +), using #12433 (upper) or Bim (C34C5) Rabbit mAb #2933 (lower).

kDa

Phospho-Bad

Bad

CIP + λ phosphatase

80

605040

30

20

10

80

605040

30

10

20

– +

Phospho-Bad (Ser136) (D25H8) Rabbit mAb #4366: WB analysis of extracts from COS-7 cells, treated with Human Epidermal Growth Factor #8916 (100 ng/ml, 30 min) in the presence or absence of calf intestinal phosphatase (CIP) plus lambda-phosphatase, using #4366 (upper) or Bad (D24A9) Rabbit mAb #9239 (lower).

Antibodies to key targets as cellular readoutsCytochrome c (D18C7) Rabbit mAb #11940: IHC analysis of paraffin-embed-ded human breast carci-noma using Cytochrome c (D18C7) Rabbit mAb in the presence of control peptide (upper) or antigen-specifc blocking peptide (lower).

Antibody sampler kits offer a convenient and economical means for the simultaneous analysis of multiple nodes within a pathway of interest.

#8678 Kit Includes: E-Cadherin (24E10) Rabbit mAb #3195, p53 (7F5) Rabbit mAb #2527, Stathmin Antibody #3352, BRCA1 Antibody #9010, Phospho-Akt (Ser473) (D9E) XP® Rabbit mAb #4060, PTEN (138G6) Rabbit mAb #9559, Phospho-Estrogen Receptor α (Ser167) (D1A3) Rabbit mAb #5587, HER2/ErbB2 (D8F12) XP® Rabbit mAb #4290, Anti-rabbit IgG, HRP-linked Antibody #7074



Phospho-Akt (Ser473) (D9E) XP® Rabbit mAb #4060: IHC analysis of PTEN heterozygous mutant mouse endometrium using #4060. (Tissue section courtesy of Dr. Sabina Signoretti, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA.)

Bak (D4E4) Rabbit mAb #12105: IHC analysis of paraffin-embedded human colon carcinoma using #12105.

Caspase-3 Activity Assay Kit #5723: NIH/3T3 cells were treated with Staurosporine #9953 (5 μM, 5 hr) and then lysed in PathScan® Sandwich ELISA Lysis Buffer (1X) #7018 (supplied with kit). Various amounts of cell lysate were added to assay plates containing the substrate solution, and plates were incubated at 37°C in the dark. Relative fluorescent units (RFUs) were acquired at 1 and 4 hr.

Assay kits to measure apoptosis

0

8000

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0 20 40 60

RFU

Lysate µg/well

Control (1 hr)Control (4 hr)Staurosporine (1 hr)Staurosporine (4 hr)

80 100

12

MDM2Ser166P

MDM2

53BP1

Ser6Ser9 Ser15

Ser20Ser37

Ser392

Bax, p53AIP, etc. Apoptosis p21Waf1/Cip1, etc. G2 arrest

p53R2, etc. DNA repair

p53P P P P

P

DNA DAMAGE

PP P

Pp53P P PP P

PPP P PP

P PPp53

Ser46 P

ATM

CAK

DNA-PK

DNA-PK

ATR

CK1δ/ε Chk2

p300

MDM2

HIPK2

PPAurora A

Degradation

Ser315

PSer315

P Ser33

p300

p53

Cytotoxic stresses, such as ionizing radiation and hypoxia can cause single- or double-strand breaks to cellular DNA. The DNA damage response (DDR) is an important cellular mechanism that prevents the cell from replicating before the damage is repaired or the cell containing damaged DNA is eliminated. The p53 tumor suppressor protein is a transcriptional regulator that plays a major role in DDR. DNA damage induces phos-phorylation of p53 at multiple sites by a number of upstream kinases including ATM, ATR, DNA-PK, and Chk2. Phosphorylation of p53 at Ser15 and Ser20 leads to a reduced interaction between p53 and its negative regulator, the oncoprotein MDM2. MDM2 inhibits p53 accumulation by targeting it for ubiquitination and proteasomal degradation. Activated p53 binds to target genes to induce a number of effector pathways including cell cycle arrest, DNA repair, senescence, or apoptosis. Studies have shown p53 to promote apoptosis through upregulation of the pro-apoptotic factors Puma and Noxa. Loss-of-function mutations in p53 or other elements of the DDR can result in cancer. In fact, up to 50% of human can-cers may contain mutations in p53 or elements of the p53 signaling pathway.

Select Reviews Wade, M, Li, YC, and Wahl, GM. (2013) Nat. Rev. Cancer 13, 83-96.v | Muller, PA and Vousden, KH. (2013) Nat. Cell Biol. 15, 2-8. Sperka, T, Wang, J, and Rudolph, KL. (2012) Nat. Rev. Mol. Cell Biol. 13, 579-590. | Delbridge, AR, Valente, LJ, and Strasser, A. (2012) Cold Spring Harb. Perspect. Biol. 4 (11).

p53 Signalingand the DNA Damage ResponseRigorously tested antibodies and controls to move your research forward

APOPTOSIS AND CANCER

kDa

Phospho-ATM (Ser1981)

ATM

200

140

100

80

200

140

100

80

– + UV

Phospho-ATM (Ser1981) (D6H9) Rabbit mAb #5883: WB analysis of extracts from 293 cells, untreated or UV-treated (100 mJ, 4 hr recovery), using #5883 (up-per) or ATM (D2E2) Rabbit mAb #2873 (lower).

Phospho-p53 (Ser15) (D4S1H) XP® Rabbit mAb (Mouse Specific) #12571: IHC analysis of paraffin-embedded 4T1 metastatic tumors in mouse lung using #12571.

Disease Connection Apoptosis is a tightly controlled process necessary for normal growth and develop-ment in multicellular organisms, playing an important role in tissue remodeling, tissue homeostasis, and wound repair. However, loss of properly regulated apoptosis can result in a host of disease states such as autoimmune diseases, neurodegenerative disorders, and cancer.

In the immune system, apoptosis plays an important role in immune tolerance and clearing T lymphocytes that recognize self-antigens. Mutations that interfere in this process can result in autoimmune disease. For example, studies have shown that loss-of-function mutations in the death receptor Fas have been linked to autoimmune lym-phoproliferative syndrome (ALPS).

Faulty apoptotic signaling can also lead to neurodegenerative disorders. In Parkinson’s disease, premature apoptosis in dopaminergic neurons can be caused by loss-of-function mutations in PINK1, a protein that normally provides protection against cytochrome c release, as well as through upregulation of extrinsic pathway death receptors. In both cases, aberrant apoptotic signaling ultimately leads to neu-ronal cell death and the patient’s eventual cognitive decline.

The misregulation of apoptosis can be most acutely associated with cancer. Suppression of apoptosis is one of the key acquired capabilities necessary for malignant trans-formation to occur. Oncogenic driver mutations found throughout the intrinsic and extrinsic pathways promote cell survival and are commonly upregulated in various forms of cancer. Although the identification of driver mutations has lead to new therapeutic opportunities, overcoming mechanisms of resistance remains a continual challenge in the field of cancer biology.

Select Reviews Renault, TT and Chipuk, JE. (2013) Ann. NY Acad. Sci.1285, 59-79. | Suzanne, M and Steller, H. (2013) Cell Death Differ. 20, 669-675. | Roulston, A, Muller, WJ, and Shore, GC. (2013) Sci. Signal. 6, pe12. | Favaloro, B, Allocati, N, Graziano V, et al. (2012) Aging 4, 330-349. | Shortt, J and Johnstone, RW. (2012) Cold Spring Harb. Perspect. Biol. 4(12).

kDa

p53

p53 siRNA II

β-Actin

200140100

80

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4030

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SignalSilence® p53 siRNA II #6562: WB analysis of extracts from HeLa cells, transfected with 100 nM Signal-Silence® Control siRNA (Fluorescein Conjugate) #6201 (-) #6562 (+), using p53 (1C12) Mouse mAb #2524 and β-Actin (13E5) Rabbit mAb #4970. p53 (1C12) Mouse mAb confirms silencing of p53 expression and β-Actin (13E5) Rabbit mAb is used to control for loading and specificity of p53 siRNA.

PathScan® Multi-Target HCA DNA Damage Kit #7101: HepG2 cells were left untreated (blue) or subjected to a 100 mJ UV treatment followed by a 1 hr recovery period (red). Mean fluorescence intensity was measured for antibodies in #7101. Data were generated on the Acumen eX3® HCS platform.

Chk2 (D9C6) XP® Rabbit mAb #6334: IHC analysis of paraffin-embedded human lung carcinoma using #6334.

Antibodies to key targets as cellular readouts for the DNA Damage Response

SignalSilence® siRNA for knockdown studies

PathScan® Multi-Target Kits for high content analysis


14

Apoptosome Formation and Activation Video

Visit www.cellsignal.com/apoptosome to view this short 3D rendered animation illustrating caspase-mediated apoptosome formation and activation from release of cytochrome c by mitochondria to prototeolytic degradation of cellular components such as actin filaments.

VALIDATION AND SUPPORT

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Saras has been with CST since 2006.

Does your antibody measure up?At CST we thoroughly validate each antibody in-house, choosing appropriate methods to verify the specificity, sensitivity and data reproducibility that you need to be confident in your results.

CST ANTIBODIES

WE DO THE RELEVANT VALIDATION, SO YOU DON’T HAVE TO...

Appropriate signal observed in all recommended applications

Clean band at appropriate molecular weight observed by western blot

Specificity confirmed by one or more of the following:

Appropriate subcellular localization Overexpression Activator or inhibitor treatment Positive and negative cell lines or tissues Phosphatase treatment RNA interference Peptide ELISA

Specific reactivity confirmed in multiple biologically relevant species and cell lines

Lot-to-lot consistency, calibrated for reliable results

Proven protocols for results you can reproduce

Competitor AbCST mAb #8822

kDa F9140100

80

6050

40

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20

miPSmES NIH

/3T3

F9 miPSmES NIH

/3T3

Seemingly comparable IF staining intensity for Nanog in F9 cells

Non-specific IF staining by competitor in Nanog-null NIH/3T3 cells

In WB, competitor recognizes multiple non-specific bands and demonstrates weak reactivity with correct bands

WB analysis reveals competitor antibody lack of specificity. IF analysis of F9 or NIH/3T3 cells using Nanog (D2A3) XP® Rabbit mAb (Mouse Specific) #8822 or a com-petitor antibody. WB analysis of various cell lines using #8822 or competitor antibody.

Are you confident that your antibody is specific? WB analysis reveals competitor antibody lack of specificity.


CST Product Scientists: Troy, (left) has been with CST since 2010, Christina (center) has been with CST since 2007, and Kenneth (right) has been with CST since 2011.

Apoptosis Signaling Pathways Poster

To request your copy of our Apoptosis Signaling Pathways poster, visit our resources page at: www.cellsignal.com/apoptosis.

Printed in the USA on 50% recycled paper (25% post-consumer waste fiber) using soy inks and processed chlorine free. © 03/2011 Cell Signaling Technology, Inc.

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Our Commitment to You As a company driven by science, our goal is to accelerate biomedical research by developing a “research tool box” that enables researchers to monitor and measure protein activity. We strive to meet contemporary and future research challenges by creating the highest quality, most specific and thoroughly validated antibodies and related reagents.

As a committed member of the research community, we practice responsible and sustainable business methods and invest heavily in research and development. We also encourage thoughtful use of our limited natural resources by highlighting environmental issues in our catalog and by promoting conservation and recycling.

All pathways were created by research scientists at Cell Signaling Technology and reviewed by leading scientists in the field. Visit www.cellsignal.com for additional reference materials and comprehensive validation data for over 3,000 antibodies and related reagents.

Apoptosis and Autophagy Pathwaysfrom Cell Signaling Technology Revised March 2011

Inhibition of Apoptosis© 2002–2011 Cell Signaling Technology, Inc.

NIK TAK1

TRAF2TRADD

FADD

PTEN

PIP3

Ras

SrcJak

Raf

PKCErk1/2

PKA

p70S6K

Bad p90RSK

CREB Stat1 Stat3

CREBStat1Stat3

Bcl-xL

Bcl-2

Cyto cApaf-1

HSP70HSP90

HSP90

HSP90

HSP27

HSP27

IKKβ

IκBNF-κB

IκB

FoxO1

FoxO1

FoxO1

Casp-3,-6,-7

Casp-9

Casp-8/10

A20CYLD

GSK-3

JNK

XIAP

NF-κB

NF-κB

Akt

Akt

PDK1

PI3K

TNFR1

TNF-α

HSP90

HSP70

IKKγ IKKβIKKα

IKKγ

cdc37

TAB

cIAP

tBidBax

Bax

Bax

FLIP

Mcl-1

Mule

Bim

Bid

Bim

Bim

HSP70

HSP27

HSP90

Smac/Diablo

[cAMP]

RIP

Survival Factors:Growth Factors,Cytokines, etc.

Apoptosis

FASBim

Bcl-2Bcl-xL

FLIPXIAPA20

Cytoplasm

Cytoplasm

Nucleus

Mitochondrial Control of Apoptosis© 2002–2011 Cell Signaling Technology, Inc.

Caspase-9

Caspase-3

Caspase-2

Bak

tBid

tBid

Bim

Bmt

Bmt

Bid

Bak

BaxBax

Bax

Bax

Bad

Bcl-2

Bcl-2

Bcl-2

Bcl-2Puma

Bcl-xL

Bcl-xL

PKC

Erk1/2

p90RSK p70 S6KAktPKA

14-3-3Bad

14-3-3

BadApaf-1

Cyto c

Arts

XIAP Smac/Diablo

AIFATM/ATR

JNK

JNK

p53

PI3K

Bim

Caspase-8,-10

FAD

D

ub

Bax

Mcl-1

Mule

PIDD

RAIDD

CaMKII

HrkDP5

HrkDP5

LC8

LC8

HSP60 HtrA2

Endo G

ING2

SirT2

Fas/CD95

FasL

Noxa

Mcl-1

Mule

Calcineurin

[NAD]

Microtubules

ApoptosisDNA Damage

Genotoxic Stress

CytosolicSequestration

Bcl-2 Family

Survival Factors:Growth Factors, Cytokines, etc.

Death Stimuli:Survival Factor Withdrawal

Pro-Apoptotic

Pro-Survival

Death Receptor Signaling © 2003–2011 Cell Signaling Technology, Inc.

FLIP

HtrA2

Gas2 Rock-1 Acinus

CAD

TNFR-1 TNFR-2 DR3APO-3

DR4/5Fas/

CD95

DA

PKTR

AD

D

TRA

DD

CYL

D

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TRA

DD

TRA

DD

RA

IDD

RIP

FasL

APO-3L/TWEAK APO-2L/

TRAIL

RIP Caspase-8,-10

ComplexIIb

Necroptosis

+zVAD

Bcl-2

Apaf-1

Cyto c

Bid

tBid

ASK1

ASK1 NIK

IKK

IκBNF-κB

NF-κB

XIAP

SmacMKK7

JNK

Lamin A Actin Fodrin ICAD PARP

Caspase-7Caspase-6

Caspase-9

Caspase-3

FAD

D

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F2

c-IA

P1/2

TRA

F2

FAD

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F2 FAD

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Dax

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ub

TNF-α TNF-α

FLIPs

RIP1 RIP3

Bcl-2

Caspase-8,-10

Cell ShrinkageMembrane Blebbing DNA Fragmentation Chromatin Condensation

DNA Repair

Apoptosis

Pro-Apoptotic

Pro-Survival

Apoptosis Overview© 2002–2011 Cell Signaling Technology, Inc.

Casp-8,-10

Casp-12

Casp-9

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Casp-3,-6,-7 Casp-9

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RIP

TRAF2

TAK1ASK1

Bid

NF-κB

NF-κB

NF-κB

IκBα

IκBα

IKKγ

IKKβIKKα

FoxO1

FoxO1

p53

p53

p53

p53 c-Jun

HtrA2

SIR2

XIAP

Arts

14-3-3

APP

CAD

CAD

ICAD

Smac/Diablo

Cyto c

Apaf-1

AIF

MuleMcl-1

Endo G

AIF Endo G

Bcl-2

Bcl-xL

tBid

Bax

BakBad

p90RSK

cdc2

ROCK ATM/ATR

Chk1/2

JNK

TNF, FasL, TRAIL

PIDDRAIDD

Puma

NoxaBim

PKC

Erk1/2

PI3K

Akt

JNK

JNK

FADD

TRAD

DRI

P1 RIP

FAD

D

TRA

DD

c-IA

P

c-IA

PTR

AF2

TRA

F2

TRADDFADD

Itch

CYLD FLIP

Calpain

ER Stress

Trophic Factors

DNA Fragmentation DNA Damage

• Cell shrinking• Membrane blebbing

Cytoplasm

Cytoplasm

Nucleus

Cell Cycle

Cellular Stress

[Ca2+]

FLIPXIAP

BimPumaNoxa

Autophagy Signaling© 2007–2011 Cell Signaling Technology, Inc. PI3K-I / Akt

SignalingAMPK

Signalingp53 / Genotoxic

Stress

PhagophoreAutophagosome

Lysosome

Lysosome

Sequestration

Fusion

ULK complex

AutophagyInduction

Autophagolysosome

Membrane Nucleation

MAPK / Erk1/2Signaling

AminoAcids

Apoptosis

RaptorGβLPRAS40

Beclin1PI3K III

LC3-I

PE

LC3

LC3-IIAtg5

Atg5

Atg12

Atg12

Atg7

Bcl-2

mTOR

p150hVPS34

Atg16

Atg16

Atg10

Atg3

Atg4

Atg7

Atg1

Apoptosis Overview Pathway Description:Apoptosis is a regulated cellular suicide mechanism characterized by nuclear condensation, cell shrink-age, membrane blebbing, and DNA fragmentation. Caspases, a family of cysteine proteases, are the central regulators of apoptosis. Initiator caspases (including caspase-2, -8, -9, -10, -11, and -12) are closely coupled to pro-apoptotic signals. Once activated, these caspases cleave and activate downstream effector caspases (including caspase-3, -6, and -7), which in turn execute apoptosis by cleaving cellular proteins following specific Asp residues. Activation of Fas and TNFR by FasL and TNF, respectively, leads to the activation of caspase-8 and -10. DNA damage induces the expression of PIDD which binds to RAIDD and caspase-2 and leads to the activation of caspase-2. Cytochrome c released from damaged mitochondria is coupled to the activation of caspase-9. XIAP inhibits caspase-3, -7, and -9. Mitochondria release multiple pro-apoptotic molecules, such as Smac/Diablo, AIF, HtrA2 and EndoG, in addition to cytochrome c. Smac/Diablo binds to XIAP which prevents it from inhibiting caspases. Caspase-11 is induced and activated by pathological proinflammatory and pro-apoptotic stimuli and leads to the activa-tion of caspase-1 to promote inflammatory response and apoptosis by directly processing caspase-3. Caspase-12 and caspase-7 are activated under ER stress conditions. Anti-apoptotic ligands including growth factors and cytokines activate Akt and p90RSK. Akt inhibits Bad by direct phosphorylation and prevents the expression of Bim by phosphorylating and inhibiting the Forkhead family of transcription factors (Fox0). Fox0 promotes apoptosis by upregulating pro-apoptotic molecules such as FasL and Bim.

Autophagy Signaling Pathway Description:Macroautophagy, often referred to as autophagy, is a catabolic process that results in the autophago-somic-lysosomal degradation of bulk cytoplasmic contents, abnormal protein aggregates, and excess or damaged organelles. Autophagy is generally activated by conditions of nutrient deprivation but has also

been associated with physiological as well as pathological processes such as development, differentia-tion, neurodegenerative diseases, stress, infection and cancer. The kinase mTOR is a critical regulator of autophagy induction, with activated mTOR (Akt and MAPK signaling) suppressing autophagy, and nega-tive regulation of mTOR (AMPK and p53 signaling) promoting it. Three related serine/threonine kinases, UNC-51-like kinase -1, -2, and -3 (ULK1, ULK2, UKL3), which play a similar role as the yeast Atg1, act downstream of the mTOR complex. ULK1 and ULK2 form a large complex with the mammalian homolog of an autophagy-related (Atg) gene product (mAtg13) and the scaffold protein FIP200 (an ortholog of yeast Atg17). Class III PI3K complex, containing hVps34, Beclin 1 (a mammalian homolog of yeast Atg6), p150 (a mammalian homolog of yeast Vps15), and Atg14-like protein (Atg14L or Barkor) or ultraviolet irradiation resistance-associated gene (UVRAG), is required for the induction of autophagy. The Atg genes control the autophagosome formation through Atg12-Atg5 and LC3-II (Atg8-II) complexes. Atg12 is conjugated to Atg5 in a ubiquitin-like reaction that requires Atg7 and Atg10 (E1 and E2-like enzymes, respectively). The Atg12–Atg5 conjugate then interacts noncovalently with Atg16 to form a large complex. LC3/Atg8 is cleaved at its C terminus by Atg4 protease to generate the cytosolic LC3-I. LC3-I is conjugated to phos-phatidylethanolamine (PE) also in a ubiquitin-like reaction that requires Atg7 and Atg3 (E1 and E2-like enzymes, respectively). The lipidated form of LC3, known as LC3-II, is attached to the autophagosome membrane. Autophagy and apoptosis are connected both positively and negatively, and extensive cross-talk exists between the two. During nutrient deficiency, autophagy functions as a pro-survival mechanism; however, excessive autophagy may lead to cell death, a process morphologically distinct from apopto-sis. Several pro-apoptotic signals, such as TNF, TRAIL, and FADD, also induce autophagy. Additionally, Bcl-2 inhibits Beclin-1-dependent autophagy, thereby functioning both as a pro-survival and as an anti-autophagic regulator.

Inhibition of Apoptosis Pathway Description:Cell survival requires the active inhibition of apoptosis, which is accomplished by inhibiting the expression of pro-apoptotic factors as well as promoting the expression of anti-apoptotic factors. The PI3K pathway, activated by many survival factors, leads to the activation of Akt, an important player in survival signaling. PTEN negatively regulates the PI3K/Akt pathway. Activated Akt inhibits the pro-apoptotic Bcl-2 family member Bad, Bax, caspase-9, GSK-3 and FoxO1 by phosphorylation. Many growth factors and cytokines induce anti-apoptotic Bcl-2 family members. The Jaks and Src phosphorylate and activate Stat3, which in turn induces the expression of Bcl-xL and Bcl-2. Erk1/2 and PKC activate p90RSK, which activates CREB and induces the expression of Bcl-xL and Bcl-2. These Bcl-2 family members protect the integrity of mitochondria, preventing cytochrome c release and the subsequent activation of caspase-9. TNF-α may activate both pro-apoptotic and anti-apoptotic pathways; TNF-α can induce apoptosis by activating caspase-8 and -10, but can also inhibit apoptosis signaling via NF-κB, which induces the expression of anti-apoptotic genes such as Bcl-2. cIAP1/2 inhibit TNF-α signaling by binding to TRAF2. FLIP inhibits the activation of caspase-8.

Death Receptor Signaling Pathway Description:Apoptosis can be induced through the activation of death receptors including Fas, TNFαR, DR3, DR4, and DR5 by their respective ligands. Death receptor ligands characteristically initiate signaling via recep-tor oligomerization, which in turn results in the recruitment of specialized adaptor proteins and activation of caspase cascades. Binding of FasL induces Fas trimerization, which recruits initiator caspase-8 via the adaptor protein FADD. Caspase-8 then oligomerizes and is activated via autocatalysis. Activated caspase-8 stimulates apoptosis via two parallel cascades: it can directly cleave and activate caspase-3, or alternatively, it can cleave Bid, a pro-apoptotic Bcl-2 family protein. Truncated Bid (tBid) translocates to mitochondria, inducing cytochrome c release, which sequentially activates caspase-9 and -3. TNF-α

and DR-3L can deliver pro- or anti-apoptotic signals. TNFαR and DR3 promote apoptosis via the adaptor proteins TRADD/FADD and the activation of caspase-8. Interaction of TNF-α with TNFαR may activate the NF-κB pathway via NIK/IKK. The activation of NF-κB induces the expression of pro-survival genes including Bcl-2 and FLIP, the latter can directly inhibit the activation of caspase-8. FasL and TNF-α may also activate JNK via ASK1/MKK7. Activation of JNK may lead to the inhibition of Bcl-2 by phosphoryla-tion. In the absence of caspase activation, stimulation of death receptors can lead to the activation of an alternative programmed cell death pathway termed necroptosis by forming complex IIb.

Mitochondrial Control of Apoptosis Pathway Description:The Bcl-2 family of proteins regulate apoptosis by controlling mitochondrial permeability. The anti-apop-totic proteins Bcl-2 and Bcl-xL reside in the outer mitochondrial wall and inhibit cytochrome c release. The proapoptotic Bcl-2 proteins Bad, Bid, Bax, and Bim may reside in the cytosol but translocate to mitochon-dria following death signaling, where they promote the release of cytochrome c. Bad translocates to mito-chondria and forms a pro-apoptotic complex with Bcl-xL. This translocation is inhibited by survival factors that induce the phosphorylation of Bad, leading to its cytosolic sequestration. Cytosolic Bid is cleaved by caspase-8 following signaling through Fas; its active fragment (tBid) translocates to mitochondria. Bax and Bim translocate to mitochondria in response to death stimuli, including survival factor withdrawal. Activated following DNA damage, p53 induces the transcription of Bax, Noxa, and PUMA. Upon release from mitochondria, cytochrome c binds to Apaf-1 and forms an activation complex with caspase-9. Although the mechanism(s) regulating mitochondrial permeability and the release of cytochrome c during apoptosis are not fully understood, Bcl-xL, Bcl-2, and Bax may influence the voltage-dependent anion channel (VDAC), which may play a role in regulating cytochrome c release. Mule/ARF-BP1 is a DNA dam-age activated E3 ubiquitin ligase for p53, and Mcl-1, an anti-apoptotic member of Bcl-2.

Direct Stimulatory Modification

Direct Inhibitory Modification

Multistep Stimulatory Modification

Multistep Inhibitory Modification

Tentative Stimulatory Modification

Tentative Inhibitory Modification

Transcriptional Stimulatory Modification

Transcriptional Inhibitory Modification GEF

GAP

Anti-apoptotic

Pro-apoptotic

Caspase

Transcription Factor GTPase

G-protein, ribosomal subunitPhosphatase

Kinase

Enzyme

ReceptorSeparation of Subunits or Cleavage Products

Joining of Subunits Translocation

At CST, providing exceptional customer service and technical support is a top priority. Our scientists work at the bench daily to produce and validate our antibodies, so they have hands-on experience and in-depth knowl-edge of each antibody’s performance. In the process, these same scien-tists generate valuable reference information that they use to answer your questions and help troubleshoot your experiment by phone or email.

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For Research Use Only. Not For Use in Diagnostic Procedures.For Research Use Only. Not For Use in Diagnostic Procedures.BROCCANAPOP_0061E

Printed on recycled paper (25% post-consumer waste fiber) using vegetable inks and processed chlorine free. © 2014 Cell Signaling Technology, Inc. Cell Signaling Technology®, SignalSilence®, PathScan®, CST™, and XP® are trademarks of Cell Signaling Technology, Inc. Acumen® is a registered trademark of TTP Labtech. DRAQ5® is a registered trademark of Biostatus Limited. DRAQ7™ is a trademark of Biostatus Limited. MitoTracker® is a registered trademark of Molecular Probes, Inc. Alexa Fluor® is a registered trademark of Molecular Probes, Inc. This product is provided under an intellectual property license from Life Technologies Corporation. The transfer of this product is contingent on the buyer using the purchased product solely in research, including use with HCS or other automated imaging applications but excluding use in combination with DNA microarrays. The buyer must not sell or otherwise transfer this product or its components for (a) diagnostic, therapeutic or prophylactic purposes; (b) testing, analysis or screening services, or information in return for compensation on a per-test basis; (c) manufacturing or quality assurance or quality control, or (d) resale, whether or not resold for use in research. For informa-tion on purchasing a license to this product for purposes other than as described above, contact Life Technologies Corporation, 5791 Van Allen Way, Carlsbad, CA 92008 USA or [email protected]

FOUNDED BY RESEARCH SCIENTISTS IN 1999, Cell Signaling Technology (CST) is a private, family-owned company with over 400 employees worldwide. Active in the field of applied systems biology research, particularly as it relates to cancer, CST understands the importance of using antibodies with high levels of specificity and lot-to-lot consistency. It’s why we produce all of our antibodies in house, and perform painstaking validations for multiple applications. And the same CST scientists who produce our antibodies also provide technical support for customers, helping them design experiments, troubleshoot, and achieve reliable results. We do this because that’s what we’d want if we were in the lab. Because, actually, we are.

CST IHC Group

FPO

Cover image: Cytochrome c release from the mitochondrion during apoptosis leads to amplification of the caspase signaling cascade via formation of the apoptosome. A radially-symmetric structure, the apoptosome assembles upon binding of cytochrome c (blue) to Apaf-1 (beige), followed by recruitment of caspase-9 (red). Once assembled, the apoptosome’s activated caspase-9 cleaves and activates cytosolic caspase-3 (red, floating) and rapidly amplifies the caspase cascade. This leads to the destruction of numerous intracellular targets by caspase-3 including the actin cytoskeleton (green).

Documents

Antibodies, Kits, and Reagents for Apoptosis Research · Staurosporine #9953: WB analysis of extracts from HeLa cells, untreated or Staurosporine-treated (3 hr), showing PARP cleavage