Co-Immunoprecipitation Assay for SIRT3 Interactors

Co-IP assay was carried out as previously reported (37 (link)). In brief, HEK293T cells transfected with plasmids expressing SIRT3-FLAG or Luc-FLAG were lysed in CHAPS lysis buffer (120 mM NaCl, 0.3% CHAPS (Sigma, C900480), 1 mM EDTA, 40 mM HEPES, pH 7.5, and cOmplete^TM protease inhibitor cocktail (Roche)) at 4°C for 2 h with rotation, and then centrifuged at 12 000 g at 4°C for 30 min to collect the supernatants. The supernatants were mixed with anti-FLAG Affinity Gel (Sigma, A2220) and rotated at 4°C overnight. The resulting immunocomplexes were washed with CHAPS lysis buffer three times, incubated with FLAG peptide in CHAPS lysis buffer at 4°C for 2 h with rotation to competitively elute SIRT3-interacting protein complexes by anti-FLAG Affinity Gel, and then centrifuged at 3 000 g at 4°C for 5 min. Finally, the resultant supernatants were boiled in 1× SDS loading buffer for 10 min and processed for western blot analysis.