Mouse polyclonal anti bcl2

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.

After evaluating the effects of direct, indirect, and combined therapy on proliferation and apoptosis, several proteins activation such as p53, Bax, Bcl-2 and β-Actin were investigated by western blotting. Half of the tumors were extracted from the animals, to determine the protein level in tissue of all groups. To perform this analysis, tissues were washed twice with PBS, then squished and combined with buffer and centrifuged for 15 minutes. The protein concentration was determined by applying the Bradford method. The proteins were dissevered by electrophoresis with sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) and then transferred onto polyvinylidene difluoride (PVDF). Finally, they were investigated with primary antibody including rabbit polyclonal anti-Bax (1:200), mouse polyclonal anti-Bcl2 (1:200), rabbit polyclonal anti-P53 proteins (1:200) (Santa Cruz Biotechnology, USA) and secondary antibodies conjugated with horse radish peroxidase (HRP) (Cell Signaling Technology, USA).the β-Actin generation signal was used as an internal control.

For western blotting analysis half of the tumors were collected from the animals. Extraction of proteins from samples were performed by RIPA buffer (10 mM Tris–HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 0.1% sodium dodecyl sulfate and 0.5% Sodium deoxycholate) containing protease and phosphatase inhibitor cocktails. Protein concentrations were examined by Bradford assay50 . Equal amounts of the total proteins were run on 12% Sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE assay) and transferred to the polyvinylidene difluoride (PVDF) and then probed with specific primary antibodies including rabbit polyclonal anti-Bax (1:200), mouse polyclonal anti-Bcl2 (1:200), rabbit polyclonal anti-P53 proteins (1:200) (Santa Cruz Biotechnology, USA) and secondary antibodies conjugated with horse radish peroxidase (HRP) (Cell Signaling Technology, USA). β-Actin was used as the control. Blots were developed by using DAB (Diaminobenzidine) staining.