Journal: eLife

Article Title: Heparan sulfates are critical regulators of the inhibitory megakaryocyte-platelet receptor G6b-B

doi: 10.7554/elife.46840

Figure Lengend Snippet: Figure 3. The heparan sulfate biosynthesis pathway is required for G6b-B binding to HEK293 cells. (A) Recombinant G6b-B, produced as a monomeric biotinylated protein and conjugated to streptavidin-PE to generate an avid probe, binds to HEL, HEK293 and COLO-320-HSR cells. (B) A genome-wide loss-of-function approach identifies the HS biosynthesis pathway as the factor required to mediate the binding of recombinant G6b-B to HEK293 cells (left panel). X- and y-axis represent the log-fold-change (LFC) and robust rank aggregation (RRA) score calculated using the MAGeCK software, respectively. Circles represent individual genes and sizes represent the false-discovery rate (FDR): large circle = FDR < 1%, small circle = 1% < FDR < 5%. Genes with FDR < 5% are color coded according to their functional annotation and genes corresponding to the HS biosynthesis pathway are additionally named. The HS biosynthesis pathway is depicted in the right panel with the genes identified in the loss-of-function approach highlighted. Similar results were obtained in HEL cells (not shown). (C) G6b-B binding to HEK293 cells was measured by flow cytometry in the presence or absence Figure 3 continued on next page

Article Snippet: DOI: https://doi.org/10.7554/eLife.46840 28 of 43 Continued Reagent type (species) or resource Designation Source or reference Identifiers Additional information Antibody anti rat IgG Alexa 546 (goat polyclonal) Invitrogen #A-11081; RRID:AB_141738 (1:300); used for IF staining of BM Antibody anti-actin (mouse monoclonal) Sigma-Aldrich #A4700, clone AC-40; RRID:AB_476730 (1:1000) Antibody Anti-a-tubulin (mouse monoclonal) Sigma-Aldrich #T6199, clone DM1A; RRID:AB_477583 (1:1000) Antibody anti-GAPDH (rabbit monoclonal) Cell Signaling Technology #2118, clone: 14C10; RRID:AB_561053 (1:10) dilution, on 0.05 mg/ml lysates for Wes Antibody anti-Src p-Tyr418 (rabbit polyclonal) Sigma-Aldrich #44660G; RRID:AB_1500523 (1:10) dilution, on 0.05 mg/ml lysates for Wes Antibody anti-Shp1 p-Tyr564 (rabbit monoclonal) Cell Signaling Technology #8849, clone: D11G5; RRID:AB_11141050 (1:10) dilution, on 0.2 mg/ml lysates for Wes Antibody anti-Shp2 p-Tyr542 (rabbit polyclonal) Cell Signaling Technology #3751; RRID:AB_330825 (1:10) dilution, on 0.2 mg/ml lysates for Wes Antibody anti-Shp2 p-Tyr580 (rabbit polyclonal) Cell Signaling Technology #3703; RRID:AB_2174962 (1:10) dilution, on 0.2 mg/ml lysates for Wes Antibody anti-Syk p-Tyr525/6 (rabbit polyclonal) Cell Signaling Technology #2711; RRID:AB_2197215 (1:50) dilution, on 0.2 mg/ml lysates for Wes Antibody anti-SH-PTP1/Shp-1 (rabbit polyclonal) Santa Cruz sc-287 (C19); RRID:AB_2173829 (1:1000) Antibody anti-SH-PTP2/Shp-2 (rabbit polyclonal) Santa Cruz sc-280 (C18); RRID:AB_632401 (1:1000) Antibody anti-phosphotyrosine (mouse monoclonal) Merck-Millipore 05–321, clone 4G10; RRID:AB_309678 (1:1000) Antibody anti-human G6b-B (mouse monoclonal) other clone 17–4 10 mg/ml, custom-made lab reagent Peptide, recombinant protein purified human IgG-Fc fragment Bethyl Laboratories P80-104 Peptide, recombinant protein recombinant Mouse Syndecan-2/CD362 protein, CF R&D Systems 6585-SD-050 Peptide, recombinant protein recombinant human Agrin protein, N-terminal, CF R&D Systems 8909-AG-050 Peptide, recombinant protein rec. human laminin 111 Biolamina LN111-02 Peptide, recombinant protein rec. human laminin 411 Biolamina LN411-02 Peptide, recombinant protein rec. human laminin 421 Biolamina LN421-02 Peptide, recombinant protein rec. human laminin 511 Biolamina LN511-02 Continued on next page Vögtle et al. eLife 2019;8:e46840.

Techniques: Binding Assay, Recombinant, Produced, Genome Wide, Software, Functional Assay, Flow Cytometry