Methods for coimmunoprecipitation and Western blot were described previously (38 (link)). Cells were harvested in 1% NP-40 lysis buffer [150 mM NaCl, 1% NP-40, 2 mM EDTA, and 50 mM tris (pH 8.0)] with protease inhibitor and phosphatase inhibitor cocktails 48 hours after transfection. Approximately 500 μg of protein was incubated overnight at 4°C with the primary antibody. Protein A beads (20 μl) were used to capture the antibody-protein complex, which was then analyzed by Western blot.
Protein samples were separated by SDS–polyacrylamide gel electrophoresis and were then transferred to a polyvinylidene difluoride membrane (Millipore). After blocking, blots were probed with the appropriate antibodies overnight at 4°C. After PBS wash, protein bands were developed using an ECL Prime Chemiluminescence kit (GE Healthcare) on a Bio-Rad developing and imaging platform.
Protein samples were separated by SDS–polyacrylamide gel electrophoresis and were then transferred to a polyvinylidene difluoride membrane (Millipore). After blocking, blots were probed with the appropriate antibodies overnight at 4°C. After PBS wash, protein bands were developed using an ECL Prime Chemiluminescence kit (GE Healthcare) on a Bio-Rad developing and imaging platform.
