Identification of the potential regulators of G6PD activity in different cell cycle

Flag-G6PD was expressed in HeLa cells. Then the HeLa cells were synchronized with a double thymidine block procedure. Briefly, the exponentially growing HeLa cells were maintained with 2 mM thymidine for 18 h, followed by a release of 9 h in fresh medium, and then cells were re-cultured in 2 mM thymidine for additional 15 h. These interphase cells were harvested. After a release of 8.5 h in fresh medium, the cells entered into M phase. Mitotic cells were harvested by mitotic shake-off. Then, cell lysate was incubated with anti-Flag M2-agarose. Elutes were separated with 10% SDS-PAGE. The gel was stained with Coomassie Brilliant Blue. Visualized bands were excised and subjected to LC-MS/MS analysis.