Bax antibody

Apigenin, Senescence Cells Histochemical Staining Kit, Griess reagent were purchased from Sigma Chemicals Co., USA. Dulbecco's modified Eagle's medium (DMEM) and Roswell Park Memorial Institute 1640 medium (RPMI-1640) supplemented with l-glutamine, fetal bovine serum (FBS), penicillin, streptomycin, Dulbecco's phosphate-buffered saline (D-PBS) and Hank's balanced salt solution (HBSS) were all procured from Gibco (Invitrogen), USA. JC-1 fluorescent dye was obtained from Life Technologies (Thermo Fisher Scientific Corporation, Carlsbad, CA, USA). Phospho-Rb (Ser780) Antibody, Bax Antibody, Bcl-2 Antibody, Anti-mouse IgG and Anti-rabbit IgG were procured from Cell Signaling Technology®, USA while Anti-p21WAF1 (Ab-1) was obtained from Calbiochem®, Darmstadt, Germany. Cyclin D1, Cyclin E, p53, p16 antibodies were procured from Santa Cruz Biotechnology, Inc., Dallas, USA while β-actin antibody was obtained from Sigma Chemicals Co., USA. JC-1 fluorescent dye was obtained from Life Technologies (Thermo Fisher Scientific Corporation, Carlsbad, CA, USA). All other chemicals used were of the highest analytical grade available. The chemicals were used as obtained without further purification. Milli-Q water obtained from Milli-Q Integral 3 system (Merck Millipore, Germany) was used for all experiments.

Cells (48 h after transfection) were washed once with PBS and lysed in RIPA buffer (Pierce; Thermo Fisher Scientific, Inc.). Protein samples were quantified with the Pierce BCA Protein assay kit (Pierce; Thermo Fisher Scientific, Inc.) and were then boiled for 10 min in sodium dodecyl sulfate (SDS) sample buffer. Equal amounts of protein (20 µg/well) were separated by 10% SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membrane. After blocking with 5% skim milk in 0.05% TBS-Tween-20 (v/v) for 1 h at room temperature, the membranes were incubated with the appropriate primary antibodies (at a dilution of 1:1,000) overnight at 4°C. Antibody specific to actin (C-2; cat no. 4967) was purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA); c-Myc (cat no. ab32072), active caspase-3 (cat no. ab2302), p27 (cat no. ab54563), p21 (cat no. ab7960) and pro-caspase-3 (cat no. ab32150) were purchased from Abcam. Bax antibody (cat no. 2772) was purchased from Cell Signaling Technology, Inc. Then membranes were washed once with TBS-Tween-20 and incubated with anti-mouse and anti-rabbit horseradish peroxidase-conjugated secondary antibodies (cat nos. ab131368 and ab191866, respectively; 1:5,000) for 2 h at room temperature. Protein bands were visualized by using WEST-ZOL (plus) Western Blot Detection system (Intron Biotechnology, Inc., Seongnam, Korea).