GPCs derived from CWRU22 and CWRU51 were sorted by FACS for CD140a at DIV160-200, directly into cell lysis buffer (NP40, Invitrogen, FNN0021) with protease inhibitor (Roche, 183617025) on ice. The insoluble fraction was removed by centrifugation at 12,000 g for 5 min at 4°C, and the supernatant analyzed for total protein with BCATM Protein Assay Kit (Thermo, 23227). 10 μg sample aliquots were separated on 4–12% gradient gels by SDS-PAGE electrophoresis (XCell SureLock, Invitrogen, 071210). Separated protein was transferred to PVDF membranes, which were blocked with 5% dry milk and incubated sequentially with a rabbit polyclonal anti-neurexin-1 antisera (Millipore, ABN161-1, 1:1000) at 4°C overnight, then washed and followed serially by a mouse monoclonal anti-β actin (Abcam, ab173838, 1:5000) at RT for 1h, and anti-mouse and anti-rabbit secondary antibodies (GE Healthcare, 95107-322 and 95107-328, 1:10000) at RT for 1h. Membranes were visualized by chemiluminescence (Mix ECLTM Reagent, GE Healthcare, RPN2236) through exposure of X-ray film. Experiments were repeated 3 times, with 3 different sets of cells.
