Ab126599

Protein was extracted using a lysis buffer (Sigma cat#C3228) containing a cocktail of phosphatase (Sigma cat#4906845001) and protease inhibitors (Sigma cat#P2714–1BTL). After homogenizing the tissues, the samples were centrifuged at 16.1 relative centrifugal force (rcf) for 15 min at 4 °C. The middle layer was transferred to a prechilled tube. Protein levels of mTOR, phosphorylated protein kinase B (PKB, also known as AKT), phosphorylated insulin receptor substrate-1 (IRS-1), PGC-1α, and glycerol kinase (GyK) were measured by immunoblotting. Total 15 μg of protein was run on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and all of the separated proteins were transferred to a nitrocellulose membrane. The membrane was blocked with 5% milk for 1 h at room temperature (RT) and exposed to the primary anti-bodies overnight at 4 °C (mTOR, Cell signaling cat#2983; pAKT, Cell signaling cat#9271; pIRS-1, Santa Cruz cat#sc17196; PGC-1α, Abcam cat#ab54481; and GyK, Abcam cat#ab126599). Then, the secondary anti-bodies were exposed for 1 h at RT. Finally, chemiluminescent substrate was applied, and bands of the protein were visualized by Molecular Imager Gel DocTM XR system. Each protein expression was normalized by glyceraldehyde 3-phosphate dehydrogenase (GAPDH), β-actin, or total AKT and quantified using ImageJ software from the NIH.