supplemental data - jbc.org · bmcrec were identified by lc-ms/ms. (c) amino acid sequences of...
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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