Rabbit polyclonal anti human bax antibody

The expression levels of Bax, Bcl-2, and GAPDH as internal control were assessed using Western blot analysis using rabbit polyclonal anti-human Bax antibody (1:1500, Abcam, Cambridge, UK), rat monoclonal anti-human Bcl-2 antibody (1:1000, Abcam, Cambridge, UK), and rabbit polyclonal anti-human GAPDH (1:1000, Abcam, Cambridge, UK), as previously described [24 (link)].

Cell lysates were extracted from cultured cells with RIPA buffer (Cell Signaling Technology) supplemented with the protease inhibitors. The lysates were subjected to ultrasonication and centrifugation. The protein concentrations were determined with the BCA Protein Assay Kit (Pierce). Equal amounts (30–40 μg) of proteins samples were separated by 8–12 % SDS–polyacrylamide gel electrophoresis and then transferred onto a nitrocellulose filter membranes (Millipore). After blocking in phosphate-buffered saline/Tween-20 containing 5 % non-fat milk, the membrane was immunoprecipitated with indicated primary antibodies overnight at 4 °C: rabbit polyclonal anti-human HIF-1α antibody (1:1000, Abcam), rabbit polyclonal anti-human Bcl-2 antibody (1:500, Abcam), rabbit polyclonal anti-human Bax antibody (1:500, Abcam), and rabbit polyclonal anti-human β-actin antibody (1:1000, Abcam). The membranes were washed with TBST (3 × 5 min), and then immunoprecipitated with the secondary antibody [HRP-conjugated goat anti-rabbit IgG antibody (Abcam)] for 1 h at room temperature. Subsequent visualization was detected with SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific). Intensities of the bands were normalized to the corresponding β-actin bands.