LC-MS/MS identification of SIRT2 interaction proteins

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.

将转染了表达Flag-Luc或Flag-SIRT2质粒的HEK293T细胞置于CHAPS裂解缓冲液（含120 mM氯化钠、0.3% CHAPS（Sigma，货号C900480）、1 mM乙二胺四乙酸（EDTA）、40 mM羟乙基哌嗪乙磺酸（HEPES，pH 7.5）以及完整蛋白酶抑制剂混合物（罗氏））中，于4℃条件下旋转裂解2小时，随后在4℃、12000 g转速下离心30分钟。针对外源性蛋白的免疫共沉淀（Co-IP）实验，将所得上清液与抗FLAG亲和凝胶（Sigma，货号A2220）混合，于4℃条件下旋转孵育过夜。通过抗FLAG亲和凝胶富集得到的SIRT2互作蛋白复合物，使用FLAG肽段进行竞争性洗脱，随后进行蛋白质印迹法（Western blotting）分析。