“…Protein extracts from H9C2 cells were prepared in RIPA homogenization buffer (20 mmol/L Tris-HCl pH 7.5, 150 mmol/L NaCl, 1 mmol/L Na 2 EDTA, 1 mmol/L EGTA, 1% Nonidet P-40), containing protease and phosphatase inhibitors. Thirty-five micrograms of protein were subjected to electrophoresis in a 10% polyacrylamide gel and transferred to a PVDF membrane and probed against specific antibodies to detect NDUFB9 (ab106699, Abcam, Cambridge, UK), COXIV (#4844, Cell Signaling Technology, Danvers, MA, USA), α-Tubulin (11H10, Cell Signaling Technology), mTOR and p-mTR (#2983 and #2974, respectively from Cell Signaling Technology), Akt and p-Akt (#9272 and #2965, respectively, from Cell Signaling Technology) as previously described [45].…”