β actin antibody

Western blots were performed as previously described [24 (link)–27 (link)]. Protein extracts were lysed in mammalian cell lysis buffer. Protein concentration was determined with the Bradford reagent (Bio-Rad Laboratories, Hercules, CA, U.S.A.) using a bovine serum albumin standard. Proteins were separated on 8–10% SDS-PAGE gels. Protein expression was determined by Western blotting using following antibodies: phospho-AKT S473, phospho-AKT T308, β-catenin, GSK3α, GSK3β, phospho-GSK-3α S21, phospho-GSK-3β S9, cyclin D1, cyclin E, mTOR, and total AKT antibodies were from Cell Signaling (Danvers, MA, U.S.A.); c-Myc, TSC1, TSC2, B-Raf, and phospho-p70S6K T421/S424 antibodies were from Epitomics (Burlingame, CA, U.S.A.); p21^Cip1, p27^Kip1, and Skp2 antibodies were from Santa Cruz (Santa Cruz, CA, U.S.A.); AKT3, phospho-mTOR S2448 and p70S6K antibodies were purchased from Millipore (Billerica, MA, U.S.A.); β-actin antibody was from Novus (Littleton, CO, U.S.A.);α-tubulin antibody was from Sigma (St. Louis, MO, U.S.A.). Anti-rabbit and anti-mouse IgG secondary antibodies were from Santa Cruz (Santa Cruz, CA, U.S.A.). α-tubulin and β-actin were used as loading control.

The enzyme-linked immunosorbent assays (ELISA) kit for TNF-α and IL-6 were from eBioscience (San Diego, CA, USA). DMEM, FBS, and all culture reagents were purchased from Gibco BRL (Life technologies, USA). Antibodies against p38, phosphorylated p38, JNK, phosphorylated JNK, ERK, phosphorylated ERK, α-tubulin, iNOS, phosphorylated NFκB p65 were purchased from Santa Cruz (Biotechnology, Inc., USA). β-actin antibody was from Novus Biologicals (Littleton, CO, USA). Antibodies against phosphorylated IκBα, phosphorylated STAT1 were purchased from Cell Signaling Technologies (Boston, MA, USA). Lipopolysaccharide (LPS, from E. coli, 0127: B8), gelatin, dexamethasone (Dex) and other chemicals were purchased from Sigma Chemicals, Co. (St Louis, Mo, USA).