Phospho pten ser380

Immunoblotting and subcellular fractionation were performed as previously described [12 (link)]. PFN1 (Abcam; ab118984, 1:2000), PTEN (Cell Signaling; 9188, 1:1000), Phospho-PTEN (Ser380/Thr382/383) (Cell Signaling; 9549, 1:1000), AKT (Cell Signaling; 4691, 1:1000), Phospho-Akt (Ser473) (Cell Signaling; 4060, 1:1000), α-Tubulin (Cell Signaling; 2144, 1:2000), SP1 (Santa cruz biotechnology; sc-59X, 1:2000), γH2AX (Abcam; ab11175, 1:2000), Chk1 (Santa cruz biotechnology, sc-377231, 1:1000), Phospho-Chk1 (Ser317) (Cell Signaling; 2344, 1:1000), Chk2 (Santa cruz biotechnology; sc-9064, 1:1000), Phospho-Chk2 (Thr68) (Novus Biologicals; NB100-92502, 1:1000), PARP (Cell Signaling; 9532, 1:1000), Cleaved-PARP(Asp214)(D64E10) (Cell Signaling; 5625, 1:1000) and β-actin (Abcam; ab54724, 1:2000), respectively followed by incubation with IgG-HRP (1:3000, Abcam, UK). Protein bands were visualized by Lumi femto solution (Dogen, Korea). Image J (National Institutes of Health, Bethesda, MD, USA) was used to quantify bands and compare to the loading control.

Heart lysates of rats were prepared in lysis buffer (20 mM Tris, 150 mM NaCl, 10% glycerol, 20 mM glycerophosphate, 1% NP40, 5 mM EDTA, 0.5 mM EGTA, 1 mM Na3VO4, 0.5 mM PMSF, 1 mM benzamidine, 1 mM DTT, 50 mM NaF, 4 μM leupeptin, pH = 8.0). Samples were resolved by 10% SDS-PAGE and transferred to PVDF membranes (Millipore). Membranes were blocked with 5% non-fat milk in TBST (50 mM Tris, 150 mM NaCl, 0.5 mM Tween-20, pH = 7.5), and then incubated with primary antibodies overnight. Antibodies used in this study were purchased from Cell Signaling Technology (CST; Danvers, MA, USA), Bioworld: total Akt (CST #4691), phospho-Akt (Ser308) (CST #4060), phospho-Akt (Thr473) (CST #13038), phospho-GSK3β (Ser9) (CST #5558), LC3A/B (CST #12741), PRAS40 (CST #2691), phospho-PRAS40 (Thr246) (CST #13175), PTEN (CST #9188), phospho-PTEN (Ser380) (CST #9551), Bax (CST2772s), Bcl2 (CST3498s), GAPDH (#AP0063), anti-rabbit IgG, (HRP-linked antibody) (CST #7074). Image J software (NIH) was used to perform densitometric analysis (http://rsb.info.nih.gov/ij/).

The following antibodies and reagents were purchased: CDK4 (#12790), CDK6 (#13331), cyclin D1 (#55506), cyclin E (#20808), p21 (#2947), p27 (#3686), PARP (#9532), caspase-9 (#9504), cleaved-caspase-3 (#9664), AKT (#4691), phospho-AKT (Ser473) (#4060), phospho-PDK1 (Ser241) (#3438), PTEN (#9188), phospho-PTEN (Ser380) (#9551) and phospho-c-raf (Ser259) (#9421) antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). Bcl-2 (ab32124), Bcl-XL (ab32370), Bax (ab32503) and cytochrome c (ab133504) antibodies were purchased from abcam. GAPDH (60004-1-Ig), Survivin (10508-1-AP), XIAP (66800-1-Ig), COXIV (11242-1-AP) and PCNA (60097-1-Ig) antibodies were purchased from Proteintech Group (Chicago, IL, USA). Rhein (R7269), Oxaliplatin (O9512) and N-acetyl-L-cysteine (NAC, A7250) were obtained from Sigma-Aldrich (St. Louis, MO, USA). DCFH-DA (S0033) and JC-1 (C2006) were purchased from Beyotime (Haimen, China). Z-DEVD-FMK (S7312), z-LEHD-FMK (S7313) and z-VAD-FMK (S8102) were purchased from Selleck (Houston, TX, USA). LY294002 (HY-10108) and 740Y-P (HY-P0175) were purchased from MCE (Monmouth, NJ, USA).