Complexome profiling of mitochondrial complex and respirasome assembly during cell cycle phase with or without NDUFA3 knockdown

HeLa cells were synchronized for interphase or mitosis. For interphase synchronization, cells were treated with 2 mM thymidine for 19 h and then harvested. For mitotic synchronization, cells were treated with 2 mM thymidine for 19 h, followed by a 9 h release in fresh medium. Mitotic cells were harvested by mitotic shake-off. Mitochondrial membrane fractions were extracted from interphase and mitotic cells using digitonin. Samples were then separated by blue native polyacrylamide gel electrophoresis (BN-PAGE). After electrophoresis, the gel was fixed and stained with Coomassie Brilliant Blue. Each entire lane was excised and cut into 13 sections from top to bottom. Each section was analyzed by LC-MS/MS for complexome profiling.