Co-Immunoprecipitation of Protein Complexes

Co-IP assays were performed as described by Zhang et al. (2014 (link)). The transfected 293 T cells were harvested and lysed with lysis buffer (1 M Tris-HCl, pH 7.4, 5 M NaCl, 0.5 M EDTA, 1% Triton X-100, and protease inhibitors). The cell lysates were centrifuged at 12,000 rpm for 5 min, and the supernatant was collected and incubated overnight with anti-FLAG M2 affinity antibody (Sigma-Aldrich, A2220) at 4°C and washed 4 times with ice-cold lysis buffer. The samples were eluted with protein loading buffer (Solarbio, P1040) and subjected to SDS-PAGE and Western blotting with anti-Myc antibody (1:1,000) or anti-Flag tag antibody (1:1,000).