Phosphorylated p iκbα

Protein of cultured cells and liver tissue was extracted using RIPA buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 1% Triton X-100, 0.5% sodium deoxycholate containing protease and phosphatase inhibitors). Protein samples (40 μg) were subjected to electrophoresis on 10% SDS-polyacrylamide gels and transferred to a nitrocellulose (Amersham Protran Supported 0.45 NC, GE healthcare). After blocking with 0.5% dry milk in PBS-Tween 0.1%, the membranes were probed with antibodies against GR (1:500, Santa Cruz Biotechnology, sc-1004), HSP90α/β (F-8) (1:1000, Santa Cruz Biotechnology, sc-13119), REV-ERBα (1:200, Santa Cruz Biotechnology, sc-100910), BMAL1 (1:500, Ripperger and Schibler, 2006 (link)), NF-κB p65 (1:200, Santa Cruz Biotechnology, sc-109), IκBα (1:200, Santa Cruz Biotechnology, sc-371), phosphorylated (p-)IκBα (1:200, Santa Cruz Biotechnology, sc-8404), tubulin (1:1000, Abcam, ab 15246) or LAMINB1 (1:500, Santa Cruz Biotechnology, sc-30264) overnight at 4°C. Anti-rabbit-IgG, mouse-IgG and goat-IgG antibody conjugated to horseradish peroxidase (HPR) was used as a secondary antibody. Detection of the immune complexes was performed using Western Bright Quantum system (Advansta) and quantification was done with the Quantity One analysis software (BioRad).

We purchased RSV, phorbol myristate acetate (PMA), 3-[4,5-dimetylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), dimethyl sulfoxide, calcium ionophore, and fetal bovine serum (FBS), from Sigma-Aldrich Corp. (St. Louis, MO, USA); TMB substrate from (Pharmingen, San Diego, CA, USA); Power SYBR^® Green PCR master mix from Applied Biosystems (Warrington, UK); penicillin/streptomycin mixture from Gibco BRL (Grand Island, NY, USA); TSLP antibodies from R&D Systems (Minneapolis, MN, USA); RIP2, caspase-1, GAPDH, phosphorylated (p)IκBα, and NF-κB p65 antibodies from Santa Cruz Biotechnology (Santa Cruz, CA, USA).