Anti irf 4 m 17

Purified murine CD4⁺ T cells were left unstimulated or stimulated as indicated in the figure legends. For detection of Tyr⁶⁹⁴STAT5 and Tyr⁶⁴¹STAT6 phosphorylation, cells were preactivated under Th9 conditions in the presence of anti-mIFNγ (5 μg ml⁻¹) and rIFN-γ (5 ng ml⁻¹) for 2 days, then washed and rested in cytokine-free medium for 8 h. Preactivated cells were treated with rat IFN-γ (5 ng ml⁻¹) in combination with either rmIL-4 (20 ng ml⁻¹) or rhIL-2 (50 U ml⁻¹) for 20 or 60 min. Whole-cell lysates were prepared as described previously47 (link), 20 μg of total protein were loaded per lane and proteins were detected according to standard protocols. The following Abs were used: anti-β-actin (AC-15, Sigma-Aldrich), anti-IRF-1 (M-20, Santa Cruz), anti-IRF-4 (M-17, Santa Cruz), anti-STAT1 (#9172, Cell Signaling) anti-P-STAT1 (Tyr⁷⁰¹, D4A7, Cell Signaling), anti-STAT5 (C-17, Santa Cruz), anti-P-STAT5 (Tyr⁶⁹⁴, C11C5, Cell Signaling), anti-STAT6 (M-20, Santa Cruz), anti-P-STAT6 (Tyr⁶⁴¹, sc11762, Santa Cruz), anti-goat IgG-HRP (#sc-2020, Santa Cruz), anti-rabbit IgG-HRP (#sc-2004, Santa Cruz), and anti-mouse IgG-HRP (#sc-2055, Santa Cruz).

For chromatin immunoprecipitation (ChIP) assays, cells were harvested and chromatin extracts were prepared using the truChIP Chromatin Shearing Reagent Kit (Covaris) according to the manufacturer’s instructions. 100 μg of the sonicated DNA–protein complexes was used for immunoprecipitions with anti-IRF4 (M-17; Santa Cruz) or normal goat Ig control (Santa Cruz) antibodies. After cross-linking was reversed and proteins were digested, the DNA was purified from the immunoprecipitates as well as from input extracts and then was analyzed by qPCR. Primer sequences are described in Supplementary Table S4.
Oligonucleotide precipitation assays (ONPs) were conducted as previously described^{19 (link)}. In brief, nuclear extracts were incubated with biotinylated double-stranded oligonucleotides corresponding to amplified IRF4-bound ChIP regions. Proteins bound to the biotin-labeled DNA were collected by streptavidin-agarose beads, separated by 10% SDS-PAGE and analyzed by immunoblot with anti-IRF4 (D9P5H; Cell Signaling) antibodies. Sequences of oligonucleotides are described in Supplementary Table S4.