Sc 1616 hrp

Forty eight hours after transfection assays, SiHa cells were harvested and protein was isolated for Western Blot assays. Briefly, the cells were washed with 1X PBS and incubated for 30 minites at 4 °C with lysis buffer containing 50 mM Tris–HCl, 150 mM NaCl, 0.5 % SDS, 1 % NP40, 0.5 mM AEBSF, 10 μg/μl antipain, 10 μg/μl aprotinin, 10 μg/μl khymostatin, 10 μg/μl leupeptin, 10 μg/μl pepstatin, 1 mM EDTA, 100 mM PMSF and 0.5 mM DTT (Sigma-Aldrich, NJ). The lysates were centrifuged at 11,000 rpm for 15 min. Total proteins from supernatants were determined using the BCA kit (Pierce, Rockford, IL). 50 μg of proteins were electrophoresed on 12 % SDS-PAGE, transferred into nitrocellulose membranes and incubated for antibodies detection. Biotinilated and pre-stained molecular weight marker was included. IgG mouse monoclonal antibody sc-7974-HRP was used to detect human PTEN protein. Human beta-actin protein was detected using IgG polyclonal antibody sc-1616-HRP (Santa Cruz, Biotechnology, Santa Cruz, CA). After the peroxidase coupled secondary goat antibody mouse anti-IgG was added, bound antibodies and protein were detected by enhanced chemiluminescence using the renaissance Western Blot kit (Pierce, Rockford, IL). The membranes were subjected to autoradiography with an intensifier screen.