Rabbit anti bax polyclonal antibody

Proteins were extracted from treated and non-treated primary T-Cell ALL patient's and Jurkat cell line using RIPA Lysis Buffer (Millipore) and following the manufacturer's instructions. pAKT, pERK Bcl-2, Bax, Pro-Caspase 3, and pCDK2 proteins were characterized in total lysates from cell cultures by Western blotting. Membranes were incubated overnight at 4° C with rabbit polyclonal anti pAKT antibody (1:200 dilution; Santa Cruz), mouse polyclonal anti pERK (1:200 dilution; Santa Cruz), mouse polyclonal anti Bcl-2 (1:500 dilution; Santa Cruz), rabbit polyclonal anti Bax antibody (1:1000 dilution; Cell Signaling), mouse polyclonal anti-Pro-Caspase 3 antibody (1:1000 dilution; Abcam), rabbit polyclonal anti pCDK2 (1:1200 dilution; Abcam). For determination of Bax and Bcl-2 ratio, antibodies against both proteins were used subsequently on the same membrane. Reactive bands were detected by chemiluminescence (Immobilon western Millipore) on a C-DiGit^® Blot Scanner (LI-COR Biosciences). A mouse polyclonal anti β-Tubulin antibody (1:1000 dilution; Sigma) was used to check for comparable protein loading and as a housekeeping protein. We performed two experiments on Jurkat cells (displayed as mean ± SD) and single experiments on each one of the 4 T-ALL patients’ samples (displayed as mean ± SD). Images were captured, stored, and analyzed using “Image studio Digits ver. 5.0” software.

Proteins (30μg) were separated by SDS-PAGE and transferred to nitrocellulose membranes. Nonspecific binding was blocked in 5% nonfat milk in Tris-buffered saline (TBS) + 0.1% Tween-20 for 1 h. The membranes were probed overnight with primary antibodies for rabbit anti-Mfn2 (Abcam, Cambridge, MA), rabbit polyclonal anti-Bcl-2 antibody (Cell Signaling, USA), rabbit polyclonal anti-Bax antibody (Cell Signaling, USA), rabbit polyclonal anti-caspase3 antibody (Cell Signaling, USA), rabbit polyclonal anti-caspase9 antibody (Cell Signaling, USA), rabbit polyclonal anti-MMP-2 antibody (Cell Signaling, USA), rabbit polyclonal anti-MMP-9 antibody (Cell Signaling, USA), rabbit polyclonal anti- integrin β1 antibody (Cell Signaling, USA) and rabbit polyclonal anti-β-actin antibody (Santa Cruz Biotech, CA) in TBS-T containing 1% nonfat milk at 4°C, washed three times with TBS-T for 10 min, and were probed with horseradish peroxidase-conjugated secondary antibodies for 1 h. The membranes were washed three times and exposed in a standard enhanced chemiluminescence reaction according to manufacturer’s instructions (Pierce, Rockford, IL.). The results were normalized to β-actin signal intensity.