Chromatin Immunoprecipitation (ChIP) was performed using commercial Protein-G coated 96 well high-throughput ChIP assay kit by loading 3 µg of cross-linked chromatin samples from HeLa cells starved overnight and then treated with IL-6 with either A) 1:50 dilution of Go-ChIP-Grade™ Purified anti-STAT3 Phospho (Ser727) Antibody (Clone A16089B), B) equal amount of Purified Mouse IgG1, κ Isotype Control Antibody (#400101), or C) competitor’s ChIP-grade Purified anti-STAT3 Phospho (Ser727) Antibody and D) equal amount of matched Isotype Control Antibody as recommended by the manufacturer. The enriched DNA was purified and quantified by real-time qPCR using primers targeting human IRF1 gene region. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the 5% of total amount of input chromatin.

Total cell lysate from Jurkat cells serum starved overnight (lane 1, 3, 5; 5 µg), or serum starved overnight and then treated with PMA (lane 2, 4, 6; 5 µg) were resolved by electrophoresis (4-12% Bis-Tris), transferred to nitrocellulose, and probed with 2µg/ml (lane 1, 2), 1µg/ml (lane 3, 4) or 0.5µg/ml (lane 5, 6) Go-ChIP-Grade7trade; Purified anti-STAT3 Phospho (Ser727) (clone A16089B) antibody. Proteins were visualized using an HRP Goat anti-mouse IgG (clone Poly40532) antibody and chemiluminescence detection. Anti-STAT3 (clone 4G4B45) antibody was used as a loading control.