Anti survivin antibody

Fetal bovine serum (FBS) and penicillin/streptomycin were obtained from Gibco/BRL Life Technologies (Grand Island, NY, USA). The anti-Bak, anti-Bax, anti-Bcl-2, anti-PARP, anti-p53, anti-phospho-p53, anti-acetyl-p53, and anti-SIRT1 antibodies were purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA). The anti-survivin antibody was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). The anti-POU3F2 antibody was from GeneTex, Inc. (Irvine, CA, USA). The anti-β-actin antibody was from Millipore Corp. (Temecula, CA, USA). The antisera to tNOX used in Western blot analyses were generated as described previously [22 (link)]. The anti-mouse and anti-rabbit IgG antibodies and other chemicals were purchased from Sigma Chemical Company (St. Louis, MO, USA) unless otherwise specified.

MB49 cells were seeded onto a six-well plate and cultured until the confluence was 80% to 90% (which occurred after approximately 24 h). At that point, UCPs/siRNA-DMNPE complex was added and the cells were incubated for different time point as indicated. Then, the cells were irradiated with or without 980 nm laser and incubated at 37°C for another 24 hours. The cells were collected using cold cell lysis reagent (Pierce) as per the manufacturer's protocol. Proteins in cell lysates were separated by SDS-PAGE on 12% acrylamide gels using a discontinuous buffer system. They were then transferred to a nitrocellulose membrane (Pierce) in transfer buffer (20 mM Tris-HCl, 190 mM glycine, 20% methanol, pH 8.3) using a Mini Trans-blot transfer system (Bio-Rad) at 100 V for 1 h. The membranes were blocked with 5% non-fat dried milk in Tris-buffered saline containing 0.05% Tween-20 (TBST) at RT for 1 h and then incubated with anti-survivin antibody (Santa Cruz, 1∶200 dilution) at RT for 1 h. After three washes in TBST, the membranes were incubated with peroxidase-conjugated anti-mouse secondary antibody (Sigma, 1∶8000 dilution) at RT for another 1 h. Three additional washes in TBST were performed before the NC membrane were visualized with ECL solution (Pierce).