Co-Immunoprecipitation and Western Blotting

Co-IP and western blotting were performed as described previously (62 (link)). Briefly, the CAB or 293T cells were seeded into 10 cm² dishes overnight, and transfected with different plasmids (a total of 10 μg) indicated on the Figure using FuGENE HD Transfection Reagent (Promega). At 24 h post-transfection, the cells were lysed by radioimmunoprecipitation (RIPA) lysis buffer with protease inhibitor cocktail (Sigma-Aldrich). After removing cellular debris, the supernatants were incubated with 15 μl anti-Flag or anti-Myc affinity gel (Sigma-Aldrich) overnight at 4°C. Immunoprecipitated proteins were resuspended in 100 μl SDS-PAGE protein loading buffer (Beyotime) after collecting by centrifugation and washing three times with lysis buffer. The whole cell lysates (WCL) or immunoprecipitated proteins were separated by 10–15% SDS-PAGE and then transferred to polyvinylidene fluoride membranes (Bio-Rad). The membranes were incubated with anti-β-actin (Cell Signaling Technology) at 1:3,000, anti-Flag/HA (Sigma-Aldrich) at 1:3,000, or anti-Myc (Santa Cruz Biotechnology) at 1:3,000, and then hybridized with the secondary HRP-conjugated anti-mouse IgG or anti-rabbit IgG (Thermo Scientific) at 1:5,000. Results are captured by using an ImageQuant LAS 4000 system (GE Healthcare) and are representative of three independent experiments.