FLAG-TRIM6 Protein Interactome Identification

pCMV-Tag2-TIRM6 or pCMV-Tag2 vector was transfected into HEK293T cells, which were harvested and extracted in the RIPA buffer 48 h later. The overexpression of FLAG-TRIM6 was confirmed by western blotting. Following a pre-clearance with IgG and protein A/G beads (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4°C for 2 h, extracts were incubated with anti-FLAG beads (Sigma-Aldrich) overnight at 4°C. The immunoprecipitated protein complexes were eluted with FLAG peptide (Sigma-Aldrich), resolved on SDS-PAGE, and stained with Coomassie Brilliant Blue. Several differently migrating bands were excised, digested with trypsin, and analyzed by LC/MS following the reported protocol (Tang et al., 2013 (link)).