Mouse anti bax

Western blotting was performed using a standard method described previously [12 (link)]. The nitrocellulose membranes were incubated with following antibodies: mouse anti-BAX (Sigma-Aldrich, St. Louis, MO, USA, Cat# WH0000581M1, RRID:AB_1840183,), mouse anti-BCL-2 (Sigma-Aldrich Cat# B3170, RRID:AB_258541), rabbit anti-phospho Akt1/2/3 (Santa Cruz Biotechnology, Dallas, TX, USA, Cat# sc-7985 also sc-7985-R, RRID:AB_667741), rabbit anti-Akt1/2/3 (Santa Cruz Biotechnology, Cat# sc-8312, RRID:AB_671714), rabbit anti-phospho-MAPK (Thermo Fisher Scientific, Waltham, MA, USA, Cat# PA1-14302, RRID:AB_1086514), rabbit anti-MAPK (Thermo Fisher Scientific Cat# PA5-14425, RRID:AB_2141578), rabbit anti-pospho JAK2 (Cell Signaling Technology, Leiden, The Netherlands, Cat# 3771S, RRID:AB_330403), rabbit anti-JAK2 (Cell Signaling Technology Cat# 4040S, RRID:AB_10691469), and mouse anti- β-Actin (Sigma-Aldrich Cat# A5316, RRID:AB_476743).

Rabbit anti-HK-2 polyclonal antibody, mouse anti-total and cleaved Caspase-3 antibodies was purchased from Cell Signaling Technology Company (USA). Rabbit anti-total and cleaved PARP antibody were purchased from Santa Cruz Biotechnology (USA). Rabbit anti-KLK10 monoclonal antibody; Mouse anti-Bax, mouse anti-Bcl-2 and mouse anti-Ki-67 monoclonal antibodies; anti-rabbit and anti-mouse horseradish peroxidase (HRP)-conjugated secondary antibodies were purchased from Sigma-Aldrich Company (St. Louis, MO, USA); HRP-linked β-actin was purchased from Shanghai KangChen Bio-tech Inc (China).

Cells were lysed in NP-40 buffer (50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% Triton X-100 and protease inhibitor cocktail (Roche)). The lysate was centrifuged at 10000×g for 20 min at 4 °C. An aliquot (10 μl) of the supernatant was subjected to electrophoresis in a 10% SDS-polyacrylamide gel, then transferred to a nitrocellulose membrane (Bio-Rad), pore size 0.2-μm, according to the method described in the manual accompanying the unit. The blots were blocked for 1 h at room temperature with 5% nonfat milk (Bio-Rad) in Tris-buffered saline, pH 8.0 (Sigma-Aldrich, St. Louis, MO). The membrane was incubated with mouse anti-Bax (1:400), mouse anti-Bcl-2 (1:400) or mouse anti-β-actin (1:3000) antibodies from Sigma-Aldrich (St. Louis, MO) in TBS-T (20 mM Tris-HCl buffer (pH 7.4) containing 150 mM NaCl and 0.05% Tween 20) overnight. Alkaline phosphatase-conjugated goat anti-mouse secondary antibodies (Sigma-Aldrich, St. Louis, MO) were added at a 1:10000 dilution in TBS-T and incubated for 1 h with slow shaking. The nitrocellulose was then washed with TBS-T (2 × 10 min) and exposed to the Sigma-Fast BCIP/NBT reagent. The intensity of the bands was quantified by densitometric analysis using Image J 1.45 software (National Institute of Health, USA).