Anti bax monoclonal antibody

Representative Western Blots were carried out in order to show CA IX, VEGF, pro-caspase 3, BAX, and BCL2 expression in normal and tumour tissue of patient with ccRCC. Human renal tissues were homogenised in RIPA buffer and incubated on ice for 1 h, and then centrifuged at 10,000g at 4 °C for 15 min. Following quantification by BCA assay kit (Thermo Fisher Scientific, Milan, Italy), 25 µg of total proteins from each supernatant were separated by SDS-PAGE under reducing conditions and then transferred to PVDF membrane. The membrane was blocked in 5% non-fat milk in 1% TBST, and then incubated overnight at 4 °C, respectively, with: 1:200 anti-CA IX monoclonal murine antibody M75²² (link), recognising N-terminal extracellular PG domain of human CA IX. 1:1000 anti-VEGF monoclonal antibody; 1:1000 anti-procaspase-3 monoclonal antibody; 1:1000 anti-BAX monoclonal antibody; 1:1000 anti-BCL2 monoclonal antibody, were all from Santa Cruz Biotechnology (Los Angeles, CA).
Membrane-bound secondary antibodies (1:12,000) (Santa Cruz Biotechnology, Los Angeles, CA) were detected by ECL following the instructions of the manufacturer (Amersham, Freiburg, Germany). To ensure equal loading amounts, blots were stripped and reprobed using a 1:2000 monoclonal antibody raised against human tubulin (Los Angeles, Santa Cruz, CA).

After the indicated treatments, total cell extracts were obtained and lysed by RIPA buffer (KeyGENBioTECH, Nanjing, China). Protein concentrations were determined according to BCA Protein Assay Kit (Beyotime, Shanghai, China). The extracted proteins in the cell lysates were isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). The primary antibodies were as follows: rabbit anti-EGFR monoclonal antibody (Cell Signaling technology, Danvers, MA, USA), anti-PTEN monoclonal antibody (Cell Signaling technology, Danvers, MA, USA), anti-p-AKT(Ser473) monoclonal antibody (Cell Signaling technology, Danvers, MA, USA), anti-AKT monoclonal antibody (Cell Signaling technology, Danvers, MA, USA), anti-cyclin D1 monoclonal antibody (Santa Cruz Biotechnology, Dallas, USA), anti-Bcl2 monoclonal antibody (Santa Cruz Biotechnology, Dallas, USA), anti-Bax monoclonal antibody (Santa Cruz Biotechnology, Dallas, USA), anti-CYP2E1 monoclonal antibody (Epitomics, CA, USA) and mouse anti-β-actin monoclonal antibody (BOSTER, Wuhan, China). Secondary antibodies include HRP-Conjugated AffiniPure Goat Anti-rabbit IgG (ZSGB-BIO, Beijing, China). Immunoreactive proteins were visualized using ECL western blotting detection regents (GE Health-care, Buckinghamshire, UK).