Goat anti mouse immunoglobulin g igg horseradish peroxidase hrp

The total proteins were harvested from the treated cells, separated on 10% sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE) gels, and then subjected to immunoblot analyses. The primary antibodies against IKKβ, Hsp70 and β-actin were purchased from Santa Cruz Biotechnology, Inc., (Santa Cruz, CA, USA); anti-IKKβ, cat# sc-8014, 1:150; anti-Hsp70, cat# sc-32239, 1:200; anti-β-actin, cat# sc-130301, 1:10,000. Secondary antibodies used in this study were goat anti-mouse immunoglobulin G (IgG)-horse radish peroxidase (HRP; cat# sc-2005, 1:5,000; Santa Cruz Biotechnology, Inc.,). Bound antibodies were detected using an enhanced chemiluminescence (ECL) system (Pierce Biotechnology, Inc., Rockford, IL, USA). The mean normalized optical density of IKKβ and Hsp70 protein bands relative to the optical density of β-actin bands from the same condition was calculated. The experiments were repeated at least three times.

ActD, LY-294002, wortmannin, deguelin, PD98059, and Hoechst 33342 were obtained from Sigma (St. Louis, MO), and SB203580 and SP600125 were obtained from Selleckchem (Houston, TX). Minimum essential medium alpha (MEMα), MEM, Dulbecco's modified Eagle medium/nutrient mixture F12 (DMEM/F12), and fetal bovine serum (FBS) were obtained from Gibco (Grand Island, NY). Goat anti-mouse immunoglobulin G (IgG)-horseradish peroxidase (HRP), and goat anti-rabbit IgG-HRP were obtained from Santa Cruz Biotech (Santa Cruz, CA). Anti-glyceraldehyde 3 phosphate dehydrogenase (GAPDH), anti-p53, and goat polyclonal secondary antibodies to mouse IgG-H&L (DyLight® 488) were obtained from Abcam (Cambridge, UK). Anti-phospho-p53 (ser15), antiphospho-HDM2 (ser166), anti-Akt and anti-phospho-Akt (Ser473) were obtained from Cell Signaling (Danvers, MA), and anti-HDM2 was obtained from Calbiochem (San Diego, CA). Serine/threonine phosphatase inhibitor cocktail was obtained from Bionovas (Toronto, Ontario). T-Pro Non-liposomal Transfection Reagent II (NTRII) was obtained from JF Ji-Feng Biotechnology (Taipei, Taiwan). Fluoromount-G was obtained from Southern Biotech (Birmingham, Alabama).