Co-Immunoprecipitation Assay Protocol

Co-IP assay was performed as previously described [16 (link)]. For transfection-based Co-IP assays, 2 × 10⁵ cells were transfected with the indicated plasmids using Lipofectamine 2000 (Invitrogen, CA, US), lysed in 500 μL of lysis buffer (50 mM Tris at pH 8.0, 500 mM NaCl, 0.5% Nonidet P-40, 1 mM dithiothreitol, and protease inhibitor tablets from Roche Applied Science), and immunoprecipitated with 20 μg anti-FLAG (Sigma-aldrich, CA, US) overnight at 4 °C. The beads were washed three times with the lysis buffer and eluted in SDS sample buffer. The eluted immunocomplexes were separated by SDS-PAGE, followed by western blotting with indicated antibodies according to the standard procedures. For detecting endogenous protein interactions, cells were lysed in 500 μL of lysis buffer and immunoprecipitated with indicated antibody or control serum (Santa Cruz Biotechnology, CA, US). After extensive washing with the lysis buffer, the immunoprecipitates were resolved by SDS-PAGE, followed by western blot analysis.