Phospho akt ser473

To collect whole-cell lysates, cells were lysed in RIPA lysis buffer supplemented with a Protease Inhibitor Cocktail (Sigma-Aldrich, St. Louis, MO, USA), and the cell lysates were centrifuged at 12,000 rpm. The supernatant was collected, mixed with loading buffer, and boiled for 15 min to disengage the protein secondary structure. The mixture was resolved on SDS-PAGE gels (BioSci™ NewFlash Protein AnyKD PAGE; DAKEWE, Beijing, China) and then transferred onto nitrocellulose membranes (GE life, Pittsburgh, PA, USA). After blocking with 5% non-fat milk, the membranes were incubated with primary antibodies overnight at 4 °C. The bands were probed with the appropriate secondary antibodies and detected by enhanced chemiluminescence. The following primary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA): PD-1 (D4W2J, 1:1000), phospho-AKT (Ser473) (D9E, 1:2000), phospho-mTOR (Ser2448) (D9C2, 1:1000), phospho-S6 Ribosomal Protein (Ser235/236) (D57.2.2E, 1:2000), AKT (pan) (C67E7, 1:1000), mTOR (7C10, 1:1000), S6 Ribosomal Protein (54D2, 1:1000), β-TrCP (D13F10, 1:1000), and β-Actin (8H10D10, 1:1000), except anti-PD-L1 antibody [EPR19759] (ab213524; Abcam, UK, 1:1000).

The protein samples were separated by SDS-PAGE gel and then electrotransferred to PVDF membranes (Millipore, USA). Subsequently, blots were subjected to immunostaining with the following primary antibodies against phospho-GSK3β (Ser9), β-actin, Beclin-1, phospho-mTOR (Ser2448), mTOR, p62 and LC3 (Cell Signaling Technology, Beverly, MA, USA); phospho-Akt (Ser473) (EPITOMICS, Burlingame, CA, USA); Akt (ABclonal Technology, ABclonal Biotech Co., Ltd., USA); and GSK3β, Atg5, FATP4 (Proteintech, China) were used at 1:1000 dilution. Goat anti-rabbit or goat anti-mouse secondary antibodies (Abbkine, Redlands, CA, USA) were used at 1:10,000 dilutions. Protein samples were obtained from cell or tissue lysates. Membrane and cytosol fraction isolation was performed according to kit instructions (Proteintech, China), and then protein concentrations were adjusted to be same among groups.

The whole-cell lysates, nuclear/cytoplasm extracts, and subcutaneous tumor lysates were prepared as described [28] (link). Then 50 µg protein samples were resolved by 10% SDS-polyacrylamide gel electrophoresis and electrophoretically transferred to polyvinylidene fluoride membranes (Millipore Corporation, Billerica, MA, U.S.). Membranes were incubated with antibodies specific for cleaved PARP (Lot No.1074-1), Akt (Lot No. 1081-1), phospho-Akt^Ser473 (Lot No. 2118-1), phospho-Akt^Thr308 (Lot No.2214-1) (Epitomics, Billerica, MA, U.S.), GRP75 (Lot No.3593S), p53 (LKtag, Shanghai, China, Lot No. 3020), phospho-p53^Ser15 (Lot No. 9284S), and phospho-p53^Ser37 (Lot No.9289) (Cell Signaling Technology, Danvers, MA, U.S.), and GAPDH (Lot No. G8795), α-tublin (Lot No. 926576) (invitrogen, Grand, NY), and LaminB1 (Abcam, USA, Lot No. ab16048) served as loading controls. Bound antibodies were visualized with Enhanced Chemiluminescence Reagent (Pierce Biotechnology Inc., Rockford, IL, U.S,).