Coimmunoprecipitation Analysis of Protein Interactions

Coimmunoprecipitation assays were performed as described previously (48 (link)). Typically, 293T cells were cotransfected with plasmids expressing GFP fusion proteins and the plasmids expressing RFP-tagged or FLAG-tagged proteins. Cells were harvested 24 h after transfection and then lysed in the immunoprecipitation assay buffer (50 mM Tris-HCl pH 7.6, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, and 1 mM phenylmethylsulfonyl fluoride). Subsequently, protein lysates were subjected to immunoprecipitation using anti-GFP magnetic beads (gtma-20, ChromoTek). After immunoprecipitation, the resultant immunoprecipitates were examined by Western blotting using specific antibodies.