Co-immunoprecipitation

Transfected cells were incubated in SF-DMEM (3 h) before treatment with or without OSM (10 ng/ml, 15 min for endogenous STAT3 or 30 min for transfected STAT3), lysis and immunoprecipitation using GFP-Trap or RFP-Trap Agarose beads (Chromotek) or Anti-FLAG M2 Magnetic beads (Sigma-Aldrich), according to the manufacturer’s instructions. Lysis and wash buffers were supplemented with PhosSTOP (Roche), cOmplete Protease Inhibitor Cocktail (Roche) and 10 mM NaF. Lysates and immunoprecipitates were analysed by SDS-PAGE and IB using antibodies against STAT3 (above), pY-STAT3 (Cell Signaling Technology, 9145), STAT1 (Cell Signaling Technology, 14994), pY-STAT1 (Tyr701, Cell Signaling Technology, 9167), FLAG (Sigma-Aldrich, F1804), GFP (Roche Applied Science, 11814460001), mCherry (Abcam, ab167453), Kα1 (Abcam, ab154399) and β-tubulin (Sigma-Aldrich, T8328), and HRP-conjugated secondary antibodies (Merck). Visualisation of bands used Western Lightning chemiluminescence reagents (PerkinElmer). Densitometric analysis was performed using Image Lab (Bio-Rad) software.