Anti bcl xl antibody

Immunoprecipitation was performed to examine the interaction of phospho-Bad and 14-3-3 interaction and Bcl-2 family proteins interaction. Protein samples (200 μg) were precleared with protein A/G agarose beads (Santa Cruz Biotechnology) to eliminate nonspecific binding proteins. They were mixed with following antibodies: anti-14-3-3, anti-Bad, and anti-Bax antibody (Santa Cruz Biotechnology) and incubated for overnight at 4°C with mild shaking. Complexes were precipitated with protein A/G agarose beads for 2 h at 4°C, washed with radioimmunoprecipitation assay buffer (Sigma Aldrich) with PMSF, and centrifuged at 10,000 g for 1 min. Supernatants were removed and remnant were boiled with sample buffer. They were loaded on 10% sodium dodecyl sulfate polyacrylamide gel, electrophoresed, and transferred to PVDF membrane. Membrane were rinsed with TBST and reacted with following antibodies: anti-phospho-Bad (1:1,000, diluted with TBST, Cell Signaling Technology), anti-Bcl-2, and anti-Bcl-xL antibody (1:1,000, diluted with TBST, Santa Cruz Biotechnology). They were washed with TBST and continuously performed as above described method in Western blot analysis.

Small intestine (100 mg) was lysed and homogenized in 1 ml lysis buffer (10 mM TBS, 1 mM EDTA, 1 mM EGTA, 2 mM sodium orthovanadate, 0.2 mM PMSF, 2 μg⁄ml leupeptin, 2 μg ⁄ml aprotinin, and 1% Triton X-100) for 30 min on ice and cleared by centrifugation at 12,000 g for 15 min at 4°C. Protein (80 μg) was fractionated on a 4 to 12% Bis-Tris gel and transferred to a 0.2-μm nitrocellulose membrane. Nitrocellulose blots were blocked by incubation in TBST (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 0.1%Tween 20) containing 5% milk for 1 h at room temperature. Western blotting was performed using the following primary antibodies at 1:1,000 dilutions: anti-Bcl-xl antibody, anti-Bcl-2 polyclonal antibody (N-19), anti-Bax antibody, anti-VEGF antibody (Santa Cruz Biotechnology), and anti-pAkt antibody (Cell Signaling). After overnight incubation with the primary antibodies at 4˚C, the membranes were washed with TBST. Immunoreactive bands were detected using HRP-linked secondary antibody (Southern Biotech, Birmingham, AL) and the Enhanced Chemiluminescence (ECL) Western blot detection kit (Amersham, Piscataway, NJ). The immunoblots were exposed to X-ray film and analyzed with the NIH Image J analysis system. Mouse anti-β-actin monoclonal antibody (1:10,000; Sigma) was used as a loading control.

Pyruvate was obtained from Sinopharm Chemical Reagent Co.Ltd (Shanghai, China) with a purity of more than 98%. 3-Methyladenine was purchased from Sigma-Aldrich (St. Louis, MO, USA). Anti-Bcl-xL antibody was obtained from Santa Cruz Biotechnology (CA, USA). The antibodies against Beclin-1, DAPK1, NMDA receptor, p-NMDA receptor, Bcl-2, Bax, caspase-3, caspase-9, cytochrome c and β-actin were purchased from Cell Signaling Technology (Boston, MA, USA). Polyclonal antibody of NMDA receptor was produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding serine 890 of human NMDAR1 [23] (link). JC-1 mitochondrial membrane potential assay kit, cell buffer for western blotting and immunoprecipitation and fluo-3/AM were purchased from Beyotime Institute of Biotechnology (Shanghai, China). SH-SY5Y cells were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). (4Z)-4-(3-Pyridylmethylene)-2-styryl-oxazol-5-one was purchased from (Merck KGaA, Darmstadt, Germany). Other reagents were obtained from Sigma-Aldrich Co. (St. Louis, MO, USA) unless indicated otherwise.