Anti bcl xl antibody

Cells were lysed in lysis buffer (250 mM NaCl, 1 mM DTT, 0.1% NP-40, 1 mM EDTA, 10 mM β-glycerophosphate, 0.1 mM Na₃VO₄H, 1 tablet protease inhibitor cocktail, 1 mM NaF, 50 mM HEPES, pH7.4, for 50 ml). Protein concentration was determined by a BCA Protein Assay Kit (Pierce). About 100 μg of protein per well was subjected to SDS-PAGE. After electrophoresis, the proteins were transferred to a nitrocellulose membrane. The transferred membranes were blocked in 5% (w/v) nonfat dry milk or 5% (w/v) BSA in TBS (0.5 M NaCl, 20 mM Tris-HCl, pH 7.4) with 0.1% (v/v) Tween 20 and probed for the first antibody, followed by incubation with a secondary antibody conjugated with horseradish peroxidase (anti-rabbit, anti-mouse; Jackson ImmunoResearch) and visualization with a detection kit (Pierce) by photographic film development. The first antibodies used in this study were as follows: anti-actin antibody (Millipore), anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and -α-tubulin antibody (GeneTex), anti-Bcl-xl antibody (Abcam), Anti-Bak,-Bax,-Bcl2,-Bim,-caspase 3,-caspase 3(A),-caspase 7(A),-Mcl-1,-poly(ADP-ribose) polymerase (PARP) and-PARP(C) antibody (Cell Signaling). Immunoblot images were quantitated by quantitative software (NIH).

The protein quality was determined by Coomassie brilliant blue staining. The protein concentrations were determined with a bicinchoninic acid (BCA) reagent assay (Beyotime, China). Forty micrograms of protein were loaded into each well of a 12% gel and subjected to SDS-PAGE. Protein was transferred to 0.45 μm nitrocellulose membranes. The membranes were blocked with 5% nonfat milk in Tris-buffered saline-Tween-20 (TBST) and then incubated with rabbit monoclonal anti-Bcl-xL antibody (1:1000 dilution, Abcam, UK), anti-Bax antibody (1:1000 dilution, Proteintech, USA) and IL-22R1 antibody (1:1000 dilution, Proteintech, USA) at 4 °C overnight. The membranes were then incubated with horseradish peroxidase-conjugated anti-rabbit secondary antibody IgG (Bioss, China) for 1 h. Next, the membranes were washed 3 times with TBST and developed via enhanced chemiluminescence (ECL) plus Western blotting detection system. β-Actin (1:1000, Santa Cruz, USA) was used as the loading control. All western blotting was performed in triplicate.