Molecular Profiling of NFATc1 and c-Jun

Antibodies for NFATc1, total-c-Jun and IκBα, as well as cyclosporine A were purchased from Santa Cruz Biotechnology (Dallas, Texas). Phospho (ser73)-c-Jun antibody was from Cell Signaling (Beverly, Massachusetts). Anti-Histone H3 (tri methyl K4) antibody was from Abcam. Antibodies for Flag and β-actin were from Sigma (St. Louis, Missouri). MG132 was from Fisher (Pittsburgh, Pennsylvania). Western blot and ChIP assays were performed as previously described (Wan et al., 2007 (link); Krzeszinski et al., 2014 (link)). NFATc1 expression plasmid was purchased from Open Biosystems (Lafayette, Colorado). Human Flag-Nur77 expression plasmid was kindly provided by Orla Conneely lab. Nur77-ΔDBD expression plasmid was constructed by deleting the amino acid residues 270–335 from the WT Nur77 expression plasmid. RNA was reverse transcribed into cDNA using an ABI High Capacity cDNA RT Kit (Life Technologies, Carlsbad, California) and analyzed using real-time quantitative PCR (SYBR Green) in triplicate. All RNA expression was normalized by the ribosomal gene L19.