Anti p s6 s240 244

Western blot analyses were performed using standard procedures using anti-P-AKT (T308) (#2965), anti-P-AKT (S473) (#9271), anti-AKT (#9272), anti-P-S6 (S240/244) (#5364), anti P-S6 (S235/236) (#4857), anti-S6 (#2217), anti-P-GSK (#9336), anti-GSK (#9322), anti-P-ERK 1/2 (#9101), anti-ERK 1/2 (#9102) (all Cell Signaling), anti GAPDH (#10R-G109a, Fitzgerald, North Acton, MA, USA) and anti-CTRP9 (#mab6537, R & D Systems, Minneapolis, MN, USA). Quantification was performed using ImageJ software version 1.52a (NIH, Bethesda, MD, USA).

The antibodies anti-pAKT Ser473 (#4060), anti-pAKT thr308 (#9275), anti-AKT (#4691), anti-pS6 S235/236 (#4858), anti-pS6 S240/244 (#5364), anti-S6, anti-pERK1/2 Thr202/Tyr204 (#9101), and anti-ERK1/2 (#4695) were from Cell Signaling Technology. Anti-LC3B was obtained from Sigma-Aldrich (L8918). KI67 was purchased from Vector laboratories (#VP K451). Anti-Actin was from MP Biomedicals (691001). Novartis Pharma AG (Basel, Switzerland) provided BYL719 and AEW541. LDE225, MK2206, AZD6482,AZD6738, GDC0032, PHA-665752, 17-AAG, LEE011, LY2835219, LBH589, ABT737 and Navitoclax were purchased from Selleckchem (Houston TX, USA). TUNEL- TREVIGEN, cat no-4815-30-K).

Anti‐H4, anti‐ac‐Tubulin, anti‐CDK1, anti‐p‐CDK1(Y15), anti‐CDK2, ‐p‐CDK2(Y15), anti‐RRM1, anti‐RRM2, and anti‐AKT (Abcam), anti‐p‐S6(S240/244), anti‐p‐CDC25C, anti‐Wee1, anti‐c‐Myc (Cell Signaling Technologies), anti‐β‐actin, anti‐PARP, anti‐Bcl‐2, anti‐Bax, anti‐Mcl‐1, anti‐ERK (Proteintech), anti‐p‐AKT (T308), anti‐p‐AKT (S473) (Affinity Biosciences), anti‐Bim, anti‐Bcl‐xL, anti‐Bak, anti‐p‐ERK(T202/Y204), anti‐CHK1 (Selleck Chemicals), anti‐ac‐H4 and anti‐γ‐H2AX (Millipore) antibodies were used for Western blot analyses.
Western blotting was performed as described previously.³³ Briefly, whole cell lysates were prepared by sonication in 10 mmol/L Tris‐Cl, pH 7.0, containing 1% SDS, protease inhibitors and phosphatase inhibitors (Roche Diagnostics). The samples were separated by electrophoresis on SDS‐polyacrylamide gels and transferred onto polyvinylidene difluoride membranes (Thermo Fisher Scientific). After blocking in TBS buffer (150 mmol/L NaCl, 10 mmol/L Tris, pH 7.4) containing 5% fat‐free milk for 1 hour at room temperature, the blots were incubated with a primary antibody overnight at 4°C and then incubated with a fluorescent‐labelled secondary antibody for 1 hour at room temperature. Immunoreactive proteins were visualized using the Odyssey Infrared Imaging System (Li‐Cor). Western blots were repeated at least three times and one representative blot is shown.