Biotinylation and Fractionation of Cell Surface Proteins

BMDMs were transferred to SFM for 30 min and then treated with 10 nM EI-tPA, 10 nM α₂M or vehicle for 1 h. Cells were washed three times with PBS, gently lifted from the plate, and then incubated with 2 mM EZ-Link Sulfo-NHS-LC-Biotin reagent (Thermo Fisher Scientific) for 30 min at 4 °C. This plasma membrane-impermeable reagent labels only the ectodomains of plasma membrane proteins. To quench biotinylation reactions, cells were extensively washed with 20 mM sodium phosphate, 150 mM NaCl, 100 mM glycine, pH 7.4. Cells were then extracted in 1% Triton X-100 containing protease inhibitors for 30 min at 4 °C. Extracts were centrifuged at 12,000× g for 20 min at 4 °C. Supernatants were collected and referred to as the Triton X-100-soluble fraction. The Triton X-100-insoluble pellets were re-extracted in RIPA buffer containing protease inhibitors for 30 min at 4 °C, then centrifuged at 12,000× g for 20 min at 4 °C. Biotinylated cell surface proteins from both fractions were affinity precipitated with Pierce Streptavidin Magnetic Beads (Thermo Fisher Scientific). Precipitates were subjected to SDS-PAGE and immunoblot analysis to detect LRP1 β-chain (Abcam), LRP1 α-chain (Sigma-Aldrich), GluN1 (Cell Signaling Technology), and PrP^C (POM19, a monoclonal antibody which is previously described^{76 (link)}). Uncropped blots are presented in Supplementary Figures online.