Protein Interaction Validation by Co-IP and WB

Interaction candidates identified by proteomics were subsequently confirmed by co-immunoprecipitation and Western blotting with antibodies against the endogenous proteins. Transfections of HEK293T cells and immunoprecipitations were carried out as described above (see “Co-immunoprecipitation and proteomics”). 1% of cell lysate (5µL/400µL) and 15% of the immunoprecipitate was loaded onto three replicate gels for analysis with multiple antibodies. Lysates and IP samples were mixed with protein loading buffer, boiled, and separated on 4–20% Tris/glycine SDS-PAGE gels (BioRad). Proteins were transferred to Immobilon-FL PVDF (Millipore), for fluorescence imaging, or nitrocellulose membranes, for chemiluminescence imaging, for 2 h at 400 mA. Immunoblots were blocked with Rockland fluorescent blocking buffer, then probed with primary antibodies at a 1:1000 dilution in the same buffer for ~2 hours at 4°C. The membrane was washed three times with TBS-T. For chemiluminescence imaging, secondary antibodies were applied at a dilution of 1:10,000 in 3% bovine serum albumin in TBS-T, then washed 3× with TBS-T prior to development with Clarity ECL Western Blotting Substrate (Bio-Rad) and imaged using a UVP ChemiDoc-It^{2 (link)} Imaging System with Visionworks Software.