SUPPLEMENTAL DATA

SUPPLEMENTAL FIGURE LEGENDS

SUPPLEMENTAL FIGURE S1. Identification of BmCREC. (A) Amino acid sequences of BmCREC show

the peptides identified in LC-MS/MS analysis (marked by red letters and rectangles) corresponding to Figure

1A. (B) Produced and purified anti-BmCREC antibody specifically detected the endogenous BmCREC (~47.5

kDa) in PSG homogenates. (C) Immunostaining of BmCREC (green) in different silkworm tissue

cryosections. The nucleus was stained with DAPI (blue). Bar, 10 µm. (D) The phylogenic tree of CREC

protein family. Five subfamilies of CREC proteins are bracketed, and the red arrow indicates BmCREC. Bt,

Bos Taurus; Ce, Caenorhabditis elegans; Dm, Drosophila melanogaster; Dr, Danio rerio; Gg, Gallus gallus;

Hm, Hydra magnipapillata; Hs, Homo sapiens; Mm, Mus musculus; Xt, Xenopus tropicalis. (E) Quantitative

real-time PCR analysis of BmCREC mRNA level in different silkworm tissues (upper panel) and in the

5th-instar PSG (lower panel). Data are shown as mean ± S.D., n = 3. (F and G) Western blotting analysis of

BmCREC in different silkworm tissues (F), and in the 5th-instar PSG (G). Br, Brain; Mal, Malpighian tubule;

Fat, fat body; ASG, anterior silkgland; MSG, middle silkgland; PSG, posterior silkgland; Mg, midgut. Days

0-7 indicate days of the 5th-instar larvae.

SUPPLEMENTAL FIGURE S2. BmCREC is an ER luminal protein. (A) The endogenous BmCREC from PSG was purified with its antibody and subjected to standard Edman degradation assay. GVPTN are shown as the first five N-terminal amino acids of endogenous BmCREC. (B) Immunostaining of BmCREC in BmN cells permeabilized with Triton X-100 (0.1%) or digitonin (25 µg/ml). Bar, 10 µm.

SUPPLEMENTAL FIGURE S3. BmCREC is associated with COPI subunits in PSG. (A) Amino acid

sequences of BmCREC and -COP show the peptides identified in LC-MS/MS analysis (marked by red

letters and rectangles) corresponding to Figure 3A. (B) GST-BmKinesin-1-CBD pull-down assay in PSG

homogenates. The precipitates were separated by SDS-PAGE and were stained with Coomassie blue. The

numbers 300 mM, 600 mM and 800 mM indicate the NaCl concentration in the elution buffer. ’-COP and

BmCREC were identified by LC-MS/MS. (C) Amino acid sequences of BmCREC and β’-COP show the

peptides identified in LC-MS/MS analysis (marked by red letters and rectangles) corresponding to (B). SUPPLEMENTAL FIGURE S4. COPI deficiency leads to accumulation of BmCREC in BmN cells. Co-immunostaining of BmCREC (red) and the cis-Golgi marker GM130 (green) in BmN cells transfected with control or β’-COP dsRNA. The corresponding DIC images are shown on the right panel. The white dash lines mark the periphery of the BmN cells. Bar, 10 µm. SUPPLEMENTAL FIGURE S5. FRET between BmCREC-CFP and KDELR-YFP in BmN cells. The

fluorescent images immediately before and after photobleaching are shown, with white rectangle areas

magnified in the lower panel. The bleaching zone is marked with a red rectangle, and the control zone is

marked with a green rectangle. FRET efficiencies (Ef) and control efficiencies (Cf) are shown in the right

columns. ***p < 0.001, n = 19, evaluated by unpaired two-tailed student’s t-test.

SUPPLEMENTAL FIGURE S6. Knockdown of BmCREC inhibits PSG tube expansion and decreases

cocoon weight in silkworm. (A) Western blotting analysis of BmCREC rescue efficiency in BmN cells. The

dsRNA-resistant GFP-BmCREC (BmCRECres) or GFP-BmCREC# (BmCREC#res, lacking HDEF) was

transfected into BmCREC dsRNA-treated cells, and the cell lysates were collected for Western blotting. (B)

Western blotting analysis of PSG homogenates from control- or BmCREC-dsRNA-injected silkworm larvae

with anti-BmCREC and anti-tubulin antibodies. (C) Quantification of the relative protein level of BmCREC to

tubulin corresponding to (B). Data are shown as mean ± S.D., ***p < 0.001, n = 10. (D) Bar graphs show the

PSG diameters. “a”, diameter of PSG lumen as shown by arrow in Figure 5I. Note that the diameters were

significantly reduced in the BmCREC-knockdown PSG. ***p < 0.001, n = 10. (E) Representative images of

the cocoons from dsRNA-injected silkworm larva. The cocoon weights are shown in the right panel. Data are

shown as mean ± S.D., **p < 0.01, n = 3, evaluated by unpaired two-tailed student’s t test.

Supplemental Figure S1

Supplemental Figure S2

Supplemental Figure S3

Supplemental Figure S4

Supplemental Figure S5

Supplemental Figure S6

Table S1: Primers used for vector construction

Primers used for cloning of silkworm gene

F-BmCREC-BglII GGG AGATCT ATGTTGAGGTTTTTCTTTGTGCTATT

R-BmCREC-XhoI TTT CTCGAG AAATTCGTCGTGCCTGGCGAGT

F-KDELR-NotI AAA GCGGCCGC ATGAATATATTCCGTTTGCTTGG

R-KDELR-XhoI AAA CTCGAG AGCTGGCAACTGTAGTTTCTTTC

F-(for sp-EGFP-BmCREC)-1 TCGATGGTGTTCGCG ATGGTGAGCAAGGGC

R-(for sp-EGFP-BmCREC)-1 GCCCTTGCTCACCAT CGCGAACACCATCGA

F-(for sp-EGFP-BmCREC)-2 GACGAGCTGTACAAG GGAGTCCCAACGAAT

R-(for sp-EGFP-BmCREC)-2 ATTCGTTGGGACTCC CTTGTACAGCTCGTC

F-(for sp-YFP-KDELR)-3 AAATCAAGATCATGTATGGTGAGCAAGGGC

R-(for sp-YFP-KDELR)-3 GCCCTTGCTCACCATACATGATCTTGATTT

F-(for sp-YFP-KDELR)-4 GACGAGCTGTACAAGGCAGGGATATCAGGA

R-(for sp-YFP-KDELR)-4 TCCTGATATCCCTGCCTTGTACAGCTCGTC

F-(for sp-CFP-BmCREC)-5 ATGTTGAGGTTTTTCTTTGTGCTATTGGCAATTTCG

ATGGTGTTCGCGATGGTGAGCAAGGGC

R-(for sp-CFP-BmCREC)-5 GCCCTTGCTCACCATCGCGAACACCATCGAAATTG

CCAATAGCACAAAGAAAAACCTCAACAT

F-(for sp-CFP-BmCREC)-6 GACGAGCTGTACAAGGGAGTCCCAACGAAT

R-(for sp-CFP-BmCREC)-6 ATTCGTTGGGACTCCCTTGTACAGCTCGTC

R-BmCREC-TAA-XhoI AAA CTCGAG TTA AAATTCGTCGTGCCTGGC

R-BmCREC#-TAA-XhoI AAA CTCGAG TTA CCTGGCGAGTGCTCCTCC

F-BmKinesin-1-CBD-EcoRI GGG GAATTC ACCCTTGCACAAAAACAAAAGATCTC

R-BmKinesin-1-CBD-SalI GGG GTCGAC TCAACTTTCATCTCTAGCGCCGAC

Table S2: Primers used for quantitative real-time PCR

F-RT-PCR-BmCREC GACCAGGCCAAAACTTTCGA

R-RT-PCR-BmCREC CGCTATTTCTCCAAGTCTCCTCTT

F-RT-PCR-RpL3 GAGGTCCCGTCGTCATCGT

R-RT-PCR-RpL3 CAGGTTTGCTAGGGTCGTCTTT

Table S3: Primers used for dsRNA synthesis

F-BmCREC-[T7]-1 [GAATTAATACGACTCACTATAGGGAGACCAC] ATG

TTG AGG TCT TTC TTT GTG CTA TT

R-BmCREC-[T7]-300 [GAATTAATACGACTCACTATAGGGAGACCAC] CTG

AGT ATA GCG GAT CCA ATC TTT C

F-β’-COPI-[T7] [GAATTAATACGACTCACTATAGGGAGACCAC] ATG

CCT CTG CGT CTG GAT ATC AA

R-β’-COPI-[T7] [GAATTAATACGACTCACTATAGGGAGACCAC] TGT

CTT ATT TTG GTA ATC CCA TAT TTT

F-EGFP-[T7]-1 [GAATTAATACGACTCACTATAGGGAGACCAC] TGG

TGA GCA AGG GCG AGG AGC

R-EGFP-[T7]-300 [GAATTAATACGACTCACTATAGGGAGACCAC] GAA

GAT GGT GCG CTC CTG GAC

F-KDELR-[T7]-1 [GAATTAATACGACTCACTATAGGGAGACCAC] ATG

AAT ATA TTC CGT TTG CT

R-KDELR-[T7]-1 [GAATTAATACGACTCACTATAGGGAGACCAC] TCA

TAG GTG GCC TTA AAC TT

F-KDELR-[T7]-2 [GAATTAATACGACTCACTATAGGGAGACCAC] CCC

ACC TTT ATC TTG GCT TT

R-KDELR-[T7]-2 [GAATTAATACGACTCACTATAGGGAGACCAC] TGC

ACA ACA CCC GAG ATA AT