P gsk3β s9

Following insulin treatment, cells were lysed in buffer containing 50 mM Tris–HCl, pH 8.0, 150 mM NaCl, 1% Triton-X-100, 1 mM EDTA, 10 mM NaF, 1 mM Na₃VO₄, and one complete protease inhibitor cocktail tablet (Roche Diagnostics)/10 ml buffer. Cell lysates were centrifuged at 15,000 × g (Eppendorf 5417R) for 10 min. The protein content of the supernatants was measured using BCA Protein Assay kit (Pierce). An equal amount of total protein was resolved on 4–20% SDS-polyacrylamide gel electrophoresis for immunoblotting analysis using standard protocols. Primary antibodies were incubated overnight at 4°C at the following dilutions, T46 (1:1000; Invitrogen), AT8 (1:500; Thermo Scientific), pGSK-3βS9, pAKT473, mTOR, P-p70S6K, P-4E-BP1, GSK-3β, AKT, anti-IRS1-pSer616 (1:500; Cell Signaling), pERK, ERK (1:500; Promega), anti-LC3 (1:400; Novus Biologicals), and anti-GAPDH (1:500, Abcam), anti-β-actin (1:5000; Sigma). Horseradish peroxidase (HRP) conjugated secondary antibodies (Amersham Biosciences) were incubated for 1 h at room temperature in 5% milk at the dilution of 1:2000 for anti-mouse and anti-rabbit antibodies. The blots were developed by ECL.

Proteins (20 μg) isolated from mouse hypothalamus were separated via 4–20% SDS-PAGE and transferred into PVDF membranes (60 min at 398 mA) using standard procedures. Blots were incubated overnight at 4°C with specific antibodies against p- AKT S473, AKT, p-GSK-3β S9, GSK-3β, p-mTOR S2448 and mTOR (Cell Signaling Technology, Denver, MA, United States) and GAPDH (Merk-Millipore, Darmstadt, Germany). Primary antibodies were diluted in PBS containing 1% albumin or 5% non-fat dry milk and 0.05% Tween. The antigen–antibody complexes were visualized using appropriate secondary antibodies (1:10 000, diluted in PBS containing 1% albumin or 5% non-fat dry milk and 0.05% Tween) and incubated for 1 h at room temperature. Blots were then extensively washed with PBS containing 0.1% Tween and developed using an enhanced chemiluminescence detection system (Pierce, Rodano, Italy). Exposition and developing time were standardized for all blots. Densitometric analysis of scanned images was performed on a Macintosh iMac computer using the public domain NIH Image J program. Results are presented as the mean ± SEM of different gels and expressed as arbitrary units (AU), which depict the ratio between levels of target phosphorylated protein and the total protein expression normalized to basal levels.

MCF10a or Ba/F3 cells stably expressing WT and mutant AKT were seeded in either 6-well plates or 60 mm dishes, and after overnight exposure to the assay medium, the cells were lysed, sonicated and 30 μg protein was loaded onto SDS-PAGE gels, transferred to nitrocellulose membranes, and immunoblotted for p-Akt and other downstream molecular targets of PI3K pathway activation. Antibodies for p-Akt (T308) (D25E6) [dilution 1:1000], p-Akt (S473) (D7F10 and D9E) [dilution 1:1000], p-PRAS40 (T246) [dilution 1:1000], p-S6RP (S240/244) [dilution 1:2000], p-GSK-3β (S9) [dilution 1:1000], and total PRAS40 [dilution 1:1000], S6RP [dilution 1:2500], GSK-3β [dilution 1:1000] were acquired from Cell Signaling Technology. V5 probe (E10) [dilution 1:2000] and β-actin antibodies (C4) [dilution 1:5000] were acquired from Santa Cruz Biotechnology.

Compounds were supplied by in-house synthesis at Genentech, Inc. or purchased from vendors. Antibodies used from immunoblots to AKT (#2920, 1:1000), AKT1 (#2938, 1:1000), AKT2 (#3063, 1:1000), AKT3 (#8018, 1:1000), pAKT(T308) (#2965, 1:1000), pAKT(S473) (#9271, 1:1000), pPRAS40(T246) (#2997, 1:1000), PRAS40 (#2691, 1:1000), pS6(S235/236) (#2211, 1:1000), S6 (#2317, 1:1000), p4EBP1 (T37/46) (#2855, 1:1000), p4EBP1 (S65) (#9456 1:1000), 4EBP1 (#9452, 1:1000), PARP (#9532 1:1000), Cleaved PARP (#5625, 1:1000), PTEN (#9556, 1:1000 and #9559, 1:1000), PIM2 (#4730, 1:500), PIM3 (#4165, 1:500), pGSK-3β (S9) (#9336, 1:500), GSK-3β (#9832, 1:500), pBAD (S112) (#9239, 1:500), and BAD (#9239, 1:500) were obtained from Cell Signaling Technology. The PIM1 antibody was obtained from Abnova (H00005292-M01, 1:500). An additional antibody to total PRAS40 was obtained from Invitrogen/ThermoFisher (AHO1031, 1:1000). Protein loading was assessed using antibodies to β-actin (Sigma-Aldrich, A5441, 1:3000), β-Tubulin (Sigma-Aldrich, T8328, 1:5000) or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Advanced ImmunoChemical, 2-RGM2, 1:2000).

Equal amounts of proteins of whole cell lysates or nuclear extracts [36 (link)] were separated by SDS-polyacrylamide gel electrophoresis. Proteins were transferred to a polyvinylidene difluoride (PVD) membrane, probed with specific antibodies, and detected using ECL detection reagent (GE Healthcare).
Antibodies against p-PDK1^S241 (#3031), p-GSK-3β^S9 (#9336), p-BAD^S136 (#39295), AKT1 (#2967), and β-catenin (#9562) were from Cell Signaling. P-Akt1^S473 (#05-736) antibody was from Upstate (Charlottesville, VA), p65 (1546-1), p-Akt1^T308 (#2214-1), and p-P70S6K^T389 (#1175-1) were supplied by Epitomics (Burlingame, CA), and c-myc (M5546) was from Sigma. The antibodies against IκBα (sc-371), P70S6K (sc-8418), BAD (sc-8044), GSK-3 (sc-7291), and topoisomerase I (TOPO I) (sc-10783) were from Santa Cruz Biotechnology (Santa Cruz, CA), PDK1 (IMG-30048) was from Imgenex (San Diego, CA), and actin (MAB1501) was from Chemicon (Temecula, CA).

Western blot analysis, cell sample harvesting, and preparation were performed by a standard procedure as presented previously [29 (link)]. We used the following primary antibodies against Nanog, β-actin (Abcam, USA), p-Akt (S473), Akt, p-Stat3 (Y705), Stat3, Erk1/2, p-Erk1/2 (T202/Y204), and p-GSK3β (S9) (all from Cell Signaling Technology, USA). Following immunodetection, each membrane was stained by amido black to confirm the transfer of the protein samples. The total level of β-actin was detected as loading control.