Immunoprecipitation and Western Blot Analysis

Immunoprecipitation methods were described previously [52 (link)]. MDA-MB-231 and HEK293T cell lysates were treated using RIPA lysis buffer supplemented with 1× inhibitor cocktail (Roche) and 1× Roche PhosSTOP phosphatase inhibitor cocktail (Roche). After centrifugation at 12,000 rpm for 15 min at 4 °C, cell debris was removed. Precleared lysates were incubated with indicated antibodies and normal IgG (as controls) overnight at 4 °C. 40 μl of protein A or protein G agarose (Santa Cruz Biotechnology, Inc.) were incubated with Lysates with rotation at 4 °C for 6 h. The complex was washed with RIPA buffer five times, resuspended with 60 μl 2× loading buffer, and cooked at 100 °C for 8 min, which was detected by western blot analysis.