Rabbit polyclonal anti bax

The total protein of INS-1 cells and mouse pancreatic islets were extracted by using a RIPA buffer. Nuclei proteins were extracted from the cells by using the Nuclear and Cytoplasmic Extraction Reagent Kit (Pierce, USA). The protein concentrations were then determined by a micro BCA assay. The total protein was separated on a 4–12% (wt/vol) SDS-PAGE. After that, the protein was transferred to a polyvinylidene fluoride (PVDF) membrane (Bio-Rad, USA). The membrane was blocked with 5% skimmed milk before being incubated overnight at 4 °C with one of the following primary antibodies: rabbit polyclonal anti-BTG₂ (Santa Cruz Biotechnology, USA), rabbit polyclonal anti-p53 (Santa Cruz Biotechnology, USA), rabbit polyclonal anti-Bax (Santa Cruz Biotechnology, USA), or mouse monoclonal anti-β-Actin (Santa Cruz Biotechnology, USA). After washing, the membrane was incubated with one of the following secondary antibodies: horseradish peroxidase-conjugated anti-rabbit IgG (Santa Cruz Biotechnology, USA), or horseradish peroxidase-conjugated anti-mouse IgG (Santa Cruz Biotechnology, USA), at room temperature. The protein bands were detected with an enhanced chemiluminescence system (Pierce Biotechnologies, USA) and exposed on x-ray films. The band intensities of proteins were quantified by using ImageJ v 1.43 software. All Western blot results were shown in supplement data.

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.