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Mapping Transmembrane Binding Partners for E-Cadherin Ectodomains

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Mapping Transmembrane Binding Partners for E-Cadherin Ectodomains SUPPLEMENTARY INFORMATION TITLE: Mapping transmembrane binding partners for E-cadherin ectodomains. AUTHORS: Omer Shafraz 1, Bin Xie 2, Soichiro Yamada 1, Sanjeevi Sivasankar 1, 2, * AFFILIATION: 1 Department of Biomedical Engineering, 2 Biophysics Graduate Group, University of California, Davis, CA 95616. *CORRESPONDING AUTHOR: Sanjeevi Sivasankar, Tel: (530)-754-0840, Email: [email protected] Figure S1: Western blots a. EC-BioID, WT and Ecad-KO cell lysates stained for Ecad and tubulin. b. HRP-streptavidin staining of biotinylated proteins eluted from streptavidin coated magnetic beads incubated with cell lysates of EC-BioID with (+) and without (-) exogenous biotin. c. C-BioID, WT and Ecad-KO cell lysates stained for Ecad and tubulin. d. HRP-streptavidin staining of biotinylated proteins eluted from streptavidin coated magnetic beads incubated with cell lysates of C-BioID with (+) and without (-) exogenous biotin. (+) Biotin (-) Biotin Sample 1 Sample 2 Sample 3 Sample 4 Sample 1 Sample 2 Sample 3 Sample 4 Percent Percent Percent Percent Percent Percent Percent Percent Gene ID Coverage Coverage Coverage Coverage Coverage Coverage Coverage Coverage CDH1 29.6 31.4 41.1 36.5 10.8 6.7 28.8 29.1 DSG2 26 14.6 45 37 0.8 1.9 1.6 18.7 CXADR 30.2 26.2 32.7 27.1 0.0 0.0 0.0 6.9 EFNB1 24.3 30.6 24 30.3 0.0 0.0 0.0 0.0 ITGA2 16.5 22.2 30.1 33.4 1.1 1.1 5.2 7.2 CDH3 21.8 9.7 20.6 25.3 1.3 1.3 0.0 0.0 ITGB1 11.8 16.7 23.9 20.3 0.0 2.9 8.5 5.8 DSC3 9.7 7.5 11.5 13.3 0.0 0.0 2.6 0.0 EPHA2 23.2 31.6 31.6 30.5 0.8 0.0 0.0 5.7 ITGB4 21.8 27.8 33.1 30.7 0.0 1.2 3.9 4.4 ITGB3 23.5 22.2 26.8 24.7 0.0 0.0 5.2 9.1 CDH6 22.8 18.1 28.6 24.3 0.0 0.0 0.0 9.1 CDH17 8.8 12.4 20.7 18.4 0.0 0.0 0.0 0.0 ITGB6 12.7 10.4 14 17.1 0.0 0.0 0.0 1.7 EPHB4 11.4 8.1 14.2 16.3 0.0 0.0 0.0 0.0 ITGB8 5 10 15 17.6 0.0 0.0 0.0 0.0 ITGB5 6.2 9.5 15.2 13.8 0.0 0.0 0.0 0.0 EPHB2 8.5 4.8 9.8 12.1 0.0 0.0 0.0 0.0 CDH24 5.9 7.2 8.3 9 0.0 0.0 0.0 0.0 Table S1: EC-BioID transmembrane protein hits. Hits that also occur in C-BioID are in bold. (+) Biotin (-) Biotin Sample 1 Sample 2 Sample 1 Sample 2 Gene ID Percent Percent Percent Percent Previously reported in coverage coverage Coverage Coverage BioID DSG2 54.2 61.1 14.3 35.3 Guo et al.1, Van Itallie et al 2 CXADR 53.4 55.3 0.0 24.9 Guo et al., Van Itallie et al CDH1 30.7 27.2 24.3 22.7 Guo et al., Van Itallie et al DSC3 22.7 23.9 4.2 9.0 Guo et al., Van Itallie et al ITGA2 18.2 18.2 0.0 0.0 EFNB1 18 18 0.0 0.0 Van Itallie et al CDH3 7 12.4 0.0 0.0 ITGB1 11.9 10.2 0.0 0.0 Guo et al. PCDH1 17 19 0.0 2.5 Guo et al. NECTIN2 44.5 38.1 0.0 0.0 Guo et al., Van Itallie et al DSC2 16.9 19.7 0.0 0.0 Van Itallie et al NECTIN3 14 18.4 0.0 0.0 CDH2 7.2 10.8 0.0 0.0 OCLN 21.7 19.2 0.0 0.0 Guo et al., Van Itallie et al NECTIN1 6 7.2 0.0 0.0 Guo et al. DSG1 8.7 4.8 11.6 12.7 Table S2: C-BioID transmembrane protein hits. Hits that also occur in EC-BioID are in bold. Experiment Binding probability (%) Total number of measured force curves Ecad:Ecad (Ca 2+) 100.0 ± 12.8 1297 Ecad:Ecad (EGTA) 56.7 ± 10.3 1134 Ecad:Pro-G (Ca 2+) 30.6 ± 7.2 1305 Sta:Ecad (Ca 2+) 38.0 ± 9.1 997 Sta:Ecad (EGTA) 12.6 ± 5.2 1060 Dsc3:Dsc3 (Ca 2+) 100.0 ± 10.4 1174 Dsc3:Dsc3 (EGTA) 34.5 ± 6.3 1225 Ecad:Dsc3 (Ca 2+) 131.1 ± 11.6 1224 Ecad:Dsc3 (EGTA) 194.4 ± 14 1224 Sta:Dsc3 (Ca 2+) 6.0 ± 2.7 1176 Sta:Dsc3 (EGTA) 19.9 ± 4.7 1274 IntA2B1:IntA2B1 (Ca 2+) 100.0 ± 14 961 IntA2B1:IntA2B1 (EGTA) 44.7 ± 8.4 1225 Ecad:IntA2B1 (Ca 2+) 94.6 ± 11.9 1265 Sta:IntA2B1 (Ca 2+) 32.8 ± 7.6 1076 c-Jun:IntA2B1 (Ca 2+) 16.3 ± 5.7 961 c-Jun:IntA2B1 (EGTA) 30.6 ± 7.7 960 EfnB1:EfnB1 (Ca 2+) 100.0 ± 15.7 1200 Ecad :EfrnB1 (Ca 2+) 298.3 ± 25.7 1310 Sta:EfrnB1 (Ca 2+) 37.6 ± 9.9 1162 Pro-G:EfrnB1 (Ca 2+) 20.6 ± 7.3 1213 CAR:CAR (Ca 2+) 100.0 ± 14.9 1206 Ecad:CAR (Ca 2+) 65.3 ± 11.1 1448 Sta:CAR (Ca 2+) 15.8 ± 6.0 1227 Pro-G:CAR (Ca 2+) 33.3 ± 10.0 923 Table S3: Measured binding probability for single molecule force measurements EC-BioID primers Forward (F) 5’-tgtgctggaattctgcagatATCCATCACACTGGCGGC Ecad 1-308 Reverse (R) 5’- tgtggatcaggctcagTGTGAGGATGCTGTAAGCG F 5’-catcctcacactgagcctgatccacatctggcgggccGGCAAGCCCATCCCCAAC V5-TurboID R 5’- cccgggcccggccCTTTTCGGCAGACCGCAG F 5’-tgccgaaaagggccgggcccgggccgacgtgtacaagcggcagATCCTCACACAAGACCCC Ecad 309-885 R 5’ -gggagatgtgttggggggaagatcTGGATCTGGATCAATGATGTTG C-BioID primers F 5’- gcgaattcgaatttaaatcgATGGGCCCTCGGTACGGC Ecad R 5’- GTCGTCCTCGCCACCTCC F 5’- atggaggtggcgaggacgacGGCAAGCCCATCCCCAAC V5-TurboID R 5’- tgctcaccatCTTTTCGGCAGACCGCAG F 5’- tgccgaaaagATGGTGAGCAAGGGCGAG EGFP R 5’ - gggagaggggcgcggccgcgTTACTTGTACAGCTCGTCCATG Table S4: List of PCR primers used in EC-BioID and C-BioID Methods: Western blot: Ecad expression in lysates of WT, Ecad-KO, EC-BioID and C-BioID cells grown on 6-well plates were tested using western blotting. Anti-Ecad (BD Biosciences) and anti- tubulin antibodies (Cell Signaling Technology) were used with anti-mouse antibody conjugated with Horse Radish Peroxidase (HRP; Invitrogen). To test for biotinylation, an identical method, as described in main text, was used for cell lysate preparation and bead incubation. After the wash step, proteins bound to beads were eluted with 25 mM biotin at 95 °C and used for western blot analysis. HRP conjugated streptavidin (Invitrogen) was used for staining of biotinylated proteins. For HRP detection WesternBright Quantum Chemiluminescence Kit (Advansta) was used. References: 1 Guo, Z. et al. E-cadherin interactome complexity and robustness resolved by quantitative proteomics. Science Signaling 7, rs7, doi:10.1126/scisignal.2005473 (2014). 2 Van Itallie, C. M. et al. Biotin ligase tagging identifies proteins proximal to E-cadherin, including lipoma preferred partner, a regulator of epithelial cell-cell and cell-substrate adhesion. Journal of Cell Science 127, 885-895, doi:10.1242/jcs.140475 (2014). Complete proteomic list obtained from MS analysis of EC-BioID Bold- Common hits in both EC-BioID and C-BioID; Italic- Transmembrane proteins in C-BioID only EC- BioID Sample 1 Sample 2 Sample 3 Sample 4 Percent Percent Percent Percent Gene ID Organism Protein Description Coverage Coverage Coverage Coverage CDH1 Canis lupus familiaris OX=9615 Cadherin 1 29.6 31.4 41.1 36.5 DSG2 Canis lupus familiaris OX=9615 Desmoglein 2 26 14.6 45 37 CXADR Canis lupus familiaris OX=9615 Coxsackievirus and adenovirus receptor 30.2 26.2 32.7 27.1 EFNB1 Canis lupus familiaris OX=9615 Ephrin B1 24.3 30.6 24 30.3 ITGA2 Canis lupus familiaris OX=9615 Integrin subunit alpha 2 16.5 22.2 30.1 33.4 CDH3 Canis lupus familiaris OX=9615 Cadherin 3 21.8 9.7 20.6 25.3 ITGB1 Canis lupus familiaris OX=9615 Integrin beta 1 11.8 16.7 23.9 20.3 DSC3 Canis lupus familiaris OX=9615 Desmocollin 3 9.7 7.5 11.5 13.3 EPHA2 Canis lupus familiaris OX=9615 EPH receptor A2 23.2 31.6 31.6 30.5 ITGB4 Canis lupus familiaris OX=9615 Integrin beta 4 21.8 27.8 33.1 30.7 ITGB3 Canis lupus familiaris OX=9615 Integrin beta 3 23.5 22.2 26.8 24.7 CDH6 Canis lupus familiaris OX=9615 Cadherin 6 22.8 18.1 28.6 24.3 CDH17 Canis lupus familiaris OX=9615 Cadherin 17 8.8 12.4 20.7 18.4 ITGB6 Canis lupus familiaris OX=9615 Integrin beta 6 12.7 10.4 14 17.1 EPHB4 Canis lupus familiaris OX=9615 EPH receptor B4 11.4 8.1 14.2 16.3 ITGB8 Canis lupus familiaris OX=9615 Integrin beta 8 5 10 15 17.6 ITGB5 Canis lupus familiaris OX=9615 Integrin beta 5 6.2 9.5 15.2 13.8 EPHB2 Canis lupus familiaris OX=9615 EPH receptor B2 8.5 4.8 9.8 12.1 CDH24 Canis lupus familiaris OX=9615 Cadherin 24 5.9 7.2 8.3 9 NENF Canis lupus familiaris OX=9615 Neudesin neurotrophic factor 70.7 70.7 69.8 69 HSP90B1 Canis lupus familiaris OX=9615 Endoplasmin 64.8 68.4 72.1 66.1 PDIA4 Canis lupus familiaris OX=9615 Protein disulfide-isomerase A4 66.2 61.7 70.9 65.4 HYOU1 Canis lupus familiaris OX=9615 Hypoxia up-regulated 1 60.9 60.8 58.3 61.8 NUCB2 Canis lupus familiaris OX=9615 Nucleobindin 2 60.4 68.3 66.8 61.3 SERPINH1 Canis lupus familiaris OX=9615 Serpin family H member 1 40.4 53.3 58.4 61 P4HB Canis lupus familiaris OX=9615 Protein disulfide-isomerase 60.4 67.1 68.2 60.6 CKAP4 Canis lupus familiaris OX=9615 Cytoskeleton associated protein 4 54.7 49.5 57.2 60.1 PC Canis lupus familiaris OX=9615 Pyruvate carboxylase 55.3 62.6 61 59.5 NUCB1 Canis lupus familiaris OX=9615 Nucleobindin 1 54.9 58.4 53 58.6 VIM Canis lupus familiaris OX=9615 Vimentin 19.1 16.3 54.7 57.5 CALU Canis lupus familiaris OX=9615 Calumenin 62.9 73.3 58.7 57.5 PRXL2A Canis lupus familiaris OX=9615 Peroxiredoxin like 2A 26.9 38 57.4 56.5 HSPA5 Canis lupus familiaris OX=9615 Heat shock protein family A (Hsp70) member 5 61.3 61.2 56.4 55.2 PDIA3 Canis lupus familiaris OX=9615 Protein disulfide-isomerase 55 50.3 56.4 54.5 DNAJC3 Canis lupus familiaris OX=9615 Uncharacterized protein 57.2 59.4 58.4 54.1 LRPAP1 Canis lupus familiaris OX=9615 LDL receptor related protein associated protein 1 56 59.9 54.1 53.3 PPIB Canis lupus familiaris OX=9615 Peptidyl-prolyl cis-trans isomerase 51.9 56.5 53.7 53.2 RCN1 Canis lupus familiaris OX=9615 Reticulocalbin 1 28.9 68.7 62.2 51.6 FAM172A Canis lupus familiaris OX=9615 Family with sequence similarity 172 member A 40.2 49.6 48.8 51.5 OS9 Canis lupus familiaris OX=9615 OS9 endoplasmic reticulum lectin 54 46.1 53.6 51.2 GOLM1 Canis lupus familiaris OX=9615 Golgi membrane protein 1 65.8 58.4 52 51 PCCA Canis lupus familiaris OX=9615 Propionyl-CoA carboxylase subunit alpha 53.1 56 54.6 48.8 SPP1 Canis lupus familiaris OX=9615 Secreted phosphoprotein 1 50.8 45.8 46.2 47.8 INHBA Canis lupus familiaris OX=9615 Inhibin subunit beta A 18.6 22.2 42.5 45.8 PDIA6 Canis lupus familiaris OX=9615
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