Phospho bad

Protein samples were extracted from cultured and treated cardiac myocytes and 40 μg of protein were subjected to SDS-PAGE (10% acrylamide) and electrotransferred onto PVDF membranes. Membranes were probed overnight at 4°C with specific primary antibodies in TTBS with 1% of BSA. CD40lg antibody was from sigma. BAD, phosphoBAD, Xiap, phosphoERK1/2 and ERK1/2 primary antibodies were from Cell Signaling. Detection was performed with the enhanced chemiluminiscence reagent ECL-plus (Amersham Bioscience) in the ImageQuant LAS 4000 mini (GE Healthcare). For quantification, the images were analyzed with “Image J” software using alpha-tubulin (Sigma) or GAPDH (Genetex) as housekeeping loading control.

Cells or exosomes were lysed in 2X Laemmli buffer (4% SDS, 20% glycerol, 10% 2-mercaptoethanol, 0.02% bromophenol blue and 120mM Tris HCl). Proteins were separated under reducing conditions in 8, 10, 12 or 15% SDS-PAGE gels, transferred onto nitrocellulose membranes, detected with AKT (pan; C67E7; 1:1000), phospho-AKT (Ser473; D9E; 1:1000), Bad (D24A9; 1:1000), phospho-Bad (Ser136/99; D25H8; 1:1000), cytochrome C (4272; 1:1000; Cell Signaling Technology, Danvers, USA), Alix (3A9; 1:500), TSG101 (4A10; 1:500; Abcam, Cambridge, UK), CD63 (Ts63; 1:500), CD81 (1.3.3.22; 1:500; Thermo Fisher, Waltham, USA), GM130 (35/GM130; 1:500; BD Biosciences, Oxford, UK), α-SMA, (1A4; 1:2000; Sigma-Aldrich, Saint Louis, USA), FN1-EDA (MAB1940; 1:2000; Merck Millipore, Burlington, USA), palladin (1E6; 1:1000; Novus Biologicals, Littleton, USA), Vimentin (Vim 3B4; 1:1000; Dako, Glostrup, Denmark) primary antibodies and horseradish peroxidase-conjugated secondary antibodies (1:4000; Dako, Glostrup, Denmark). Specific signal was visualized using the SuperSignal West Dura Chemi-luminescent detection kit (Thermo Scientific, Waltham, USA). Membranes were probed for β-actin (C4; 1:5000; BD Biosciences, San Jose, USA), HSP90 (1:1000; Cell Signaling Technology, Danvers, USA) or HSC-70, (B-6; 1:2000; Santa Cruz Biotechnology, Dallas, USA), as loading controls.

N-nitrosomethylbenzylamine (NMBA) was purchased from Ash Stevens Inc. (Detroit, MI, USA). α-Tocopherol (α-T) with purities of 95.5% was purchased from Sigma Aldrich (St. Louis, MO, USA). in vitro studies, α-Tocopherol was purified by flash chromatography to purities > 99%. Primary antibodies against PCNA, PPARγ, PTEN, phospho-Akt (Ser473), phospho-mdm2 (Ser166), phospho-p53 (Ser15), p21 Waf1/Cip1, phospho-Bad (Ser136), Bax, Bcl-2, phospho-cdc2, phospho-cdc25C, and HRP-labeled secondary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies for immunohistochemistry against phospho-Akt, PPARγ, and p53 were purchased from Merck KGaA (Darmstadt, Germany) or LifeSpan BioSciences. Inc. (Seattle, USA). The HRP-labeled anti-β-actin antibody was from Sigma-Aldrich (St. Louis, MO, USA). The PPARG-specific agonist Rosiglitazone (RGZ) and antagonist GW9662 (GW) were purchased from Selleck Chemicals (Houston, TX, USA).

Ubenimex was provided by Shenzhen Main Luck Pharmaceuticals Inc. (Shenzhen, China). 5-fluorouracil was purchased from Xudong Haipu pharmaceutical Co., LTD. (Shanghai, China). Cisplatin and Oxaliplatin were obtained from Qilu Pharmaceutical Co., Ltd. (Jinan, China). Primary antibodies against human Pim-3, BCL-XL, BCL-2, Bad, and phospho-Bad were purchased from Cell Signaling Technology (Beverly, MA). Antibodies against human CD13, β-actin, P-gp, MRP2, MRP3 and ABCG2 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Horseradish Peroxidase (HRP)-conjugated Affini-pure Goat Anti-Rabbit IgG (H+L) was acquired from Proteintech (Chicago, USA).

Dulbecco’s modified Eagle’s medium was obtained from Life Technologies (Grand Island, NY, USA); antibodies against caspases, phospho-ERK1/2, phospho-JNK, phospho-p38, Akt, phospho-Akt, phospho-Bad, HER2 and phospho-HER2 were obtained from Cell Signaling (Beverly, MA, USA); and those against c-Jun, survivin, XIAP, Bcl-2 and PARP were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Resveratrol was purchased from Calbiochem (San Diego, CA, USA). All other chemicals including docetaxel were purchased from Sigma Chemicals (St. Louis, MO, USA).

Radiation treatment was performed with a 6 MV-X ray source (Varian Medical Systems, California, USA), and the cells were irradiated at a dose rate of 0.2 Gy/min. PKI-587 (MeDeCm Express, USA) was dissolved in dimethyl sulfoxide at 2 mM and stored at -20°C. Antibodies to Ki67, PI3Kp110α, PI3Kp110γ, Akt, phospho-Akt (Ser473), mTOR, phospho-mTOR (Ser2448), Bad, phospho-Bad, Caspase-3, Cleaved Caspase-3, Caspase-9, Cleaved Caspase-9, PARP, and Cleaved RARP were purchased from Cell Signaling Technology, USA. Antibodies to phospho-Rb, Rb, Cyclin D1, phospho-DNAPKcs (Ser2056), DNAPKcs, phospho-ATM (Ser1981), ATM, phospho-ATR (Ser428), and ATR were acquired from Abcam, USA. Antibodies to p70S6K, phospho-S6K1 (T421 + S424), eIF4EBP1 and phospho-eIF4EBP1 were purchased from Boster Biological Technology, China. γ-H2AX was obtained from Biolegend Biological Technology, USA. β-actin was obtained from Biosharp Life Science, China. SP-9000 was obtained from Zhongshan Gold Bridge.