HEK293T cells transfected with plasmids expressing Flag-Luc or Flag-SIRT2 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 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. For Co-IP with exogenous proteins, the resulting supernatants were mixed with anti-FLAG Affinity Gel (Sigma, A2220) and rotated overnight at 4 °C. The SIRT2 interacting protein complexes by anti-Flag Affinity Gel were competitively eluted using FLAG peptides and processed for Western blotting analysis.