Anti cyclin d1 monoclonal antibody

Samples were separated by 10% SDS-polyacrylamide gel electrophoresis, followed by transfer to PVDF membrane. After blocking in phosphate-buffered saline containing 5% bovine serum albumin and 0.1% Tween-20, the membrane was incubated with anti-myc monoclonal antibody (Cell Signaling Technology Inc.), anti-HA monoclonal antibody (Cell Signaling Technology Inc.), anti-Cyclin D1 monoclonal antibody (Santa Cruz Inc.), anti-PPIL3 polyclonal antibody (Abcam) or anti-β-actin monoclonal antibody (Sigma) at room temperature for 2 h, followed by incubation with a peroxidase-linked secondary antibody (CalbioChem) at room temperature for one h. The signals were detected using Western blotting Luminol Reagent (Santa Cruz Inc.).

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).