Phospho akt thr308 antibody

The CCK8 kit was purchased from MedChemExpress in the United States, the transwell cell was purchased from Corning in the United States, and the Annexin V-FITC/PI apoptosis kit was purchased from Sigma-Aldrich in the United States. Phospho-Akt (Ser473) antibody, Phospho-Akt (Thr308) antibody, Akt (pan) antibody, mTOR antibody, and Phospho-mTOR (Ser2448) antibody were purchased from Cell Signaling Technology, USA.

For Western Blot Analysis, the cells were lysed using the mammalian protein extraction reagent RIPA Lysis Buffer (Beyotime, Haimen, China) supplemented with a protease inhibitor cocktail (Roche, Mannheim, Germany) or 1%PMSF(Wuhan, China) and then clarified by centrifugation. Protein concentration was quantified using the BCA Protein Assay Kit (Pierce). The proteins (30 μg or 50 μg) were electrophoresed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto PVDF membranes. Immunoblotting was performed using the human anti-G protein alpha inhibitor 2 antibody (ab20392, 1:500; abcam, UK), the Phospho-AKT (Thr308) Antibody (9275, 1:1,000; Cell Signaling, USA), the Phospho-AKT (Ser473) Antibody (9271, 4060, 1:1,000; Cell Signaling, USA), the AKT antibody (9292, 1:1,000; Cell Signaling, USA), and the PTEN Antibody (9552, 1:1,000; Cell Signaling, USA); The GAPDH (14C10) Rabbit mAb (2118, 1:1,000; Cell Signaling, USA), the β-Actin (13E5) Rabbit mAb (4970, 1:1,000; Cell Signaling, USA), and the β-Tubulin (9F3) Rabbit mAb (2128, 1:1,000; Cell Signaling, USA) were used as internal control proteins. Chemiluminescent signal was detected by ECL staining (Cwbiotech, Beijing, China).