Phosphorylation Dynamics of STAT Proteins

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).