Anti histone h3 tri methyl k4 antibody

Protein samples were mixed with Laemmli buffer (BioRad) and β-mercaptoethanol (Sigma). Each sample was analyzed in triplicate. Next, samples were denatured, loaded on a gel (0.1 µg protein per sample), and separated by electrophoresis (60 min, 125 V). Then proteins were blotted onto a polyvinylidene difluoride (PVDF) membrane using a Trans-Blot Turbo Transfer System (BioRad). Membranes were blocked with 3% BSA for 1 h at RT and incubated with primary antibodies (anti-histone H3 (tri methyl K9) antibody (Abcam), anti-histone H4 (tri methyl K20) antibody (Abcam), anti-histone H4 (acetyl K8) antibody (Abcam), anti-histone H3 (tri methyl K4) antibody (Abcam), anti-histone H3 (tri methyl K27) antibody (Abcam), and anti-histone H3 (acetyl K9) antibody (Abcam)) in TBST for 16 h at 4 °C. The membranes were then washed three times for 10 min with TBST, and were incubated with secondary antibody (anti-rabbit) for 1 h at RT and washed. The chemiluminescence reaction was performed with Clarity Western ECL Substrate (BioRad). Signals were visualized with ChemiDoc Imaging System (BioRad), and signal intensities were measured using ImageLab software (BioRad).

ChIP was performed using anti-histone H3 (tri methyl K4) antibody (Abcam) and anti-acetyl-histone H3 antibody (Millipore). The amount of immunoprecipitated chromatin was determined by qPCR analysis as previously described^{41 (link)}. The primer pairs used for ChIP-qPCR covered 3–177 bp of the SOC1 coding region (Supplementary Table 3).

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.