Psgl 1 fc

The anti-EV-A71 mAbs MA28-7 (mouse IgG₁) [11 (link)] and MA105 (mouse IgG_2b) [23 (link)], were generated from mice immunized with EV-A71-1095. The anti-EV-A71 mAb 10F0 (IgG₁) was purchased from Abcam. Anti-DAF antibody (IF7, mouse IgG_2b) [34 (link)] was used to inhibit Coxsackievirus B3 binding to HeLa cells. For negative controls, mouse IgG₁ (MOPC-21) and IgG_2b (MOPC-141) were purchased from Biolegend and Sigma-Aldrich, respectively. Soluble recombinant forms of human proteins fused to the Fc region of human IgG₁ (PSGL-1-Fc, SCARB2-Fc, and CTLA-4-Fc) were purchased from R&D Systems. CTLA-4-Fc was used as a negative control Fc protein.

2 μg/ml recombinant P-selectin-Fc and PSGL-1-Fc (R&D Systems) were co-incubated for 2 h in PBS plus 1 mm CaCl₂ for the cell-free assay or platelet lysates were obtained using immunoprecipitation lysis buffer (150 mm NaCl, 0.5% Nonidet P-40, 10 mm Tris, pH 7.3). The cell-free experiments were performed using P-selectin-Fc and PSGL-1-Fc (recombinant fusion proteins of P-selectin and PSGL-1 with immunoglobulin crystallizable fragments (Fc)) to allow efficient resuspension of P-selectin and PSGL-1. This is a common strategy to analyze membrane proteins that have low hydrophilicity (30 (link), 31 (link)). Where indicated, 5 μm full-length Efb or Efb-N was incubated with the protein suspensions/platelet lysates. Either P-selectin or PSGL-1 (10 μg/ml) was immunoprecipitated using specific antibodies. Immunoprecipitated and co-immunoprecipitated proteins were identified by immunoblotting.

NS0-expressed ADAM10, ADAM17, MMP1, 2, 7, 8, 9, 12, 13, and 14 were purchased from R & D Systems (Oxon, United Kingdom). They were diluted to a concentration of 100 µg/mL in assay buffer (50 mM Tris, 10 mM CaCl₂, 150 mM NaCl, 0.05% Brij-35 (w/v), pH 7.5, for MMP14 supplemented with 5 µM ZnCl₂) and stored at −80 °C in aliquots until further use. Recombinant human IL-8 (CXCL8, 72 aa and 77 aa) was purchased from PeproTech. All chemokines were diluted to 100 µg/mL in PBS and stored at −20 °C. Mca-K-P-L-G-L-Dpa-A-R-NH2 Fluorogenic Peptide Substrate IX (R & D Systems) was stored as 2 mM in dimethyl sulfoxide (DMSO) at −80 °C. PSGL-1-Fc was purchased from R & D Systems.