Evaluation of potential inhibitory activity of Stat5b^m/m on protease activity.

Recombinant FLAG-Stat5b was incubated for 4 hours at 37°C with 35 μg CHAPS extract from FdCP1 cells, either with (lane 3) or without (lane 2) 0.5 mmol/L phenylmethylsulfonyl fluoride. Increasing amounts of either recombinant Stat5b^m/m (lanes 4-6) or recombinant Stat5b^WT (lanes 7-9) were added to the reaction in ratios of 1:1 (lanes 4, 7), 2:1 (lanes 5, 8), or 8:1 (lanes 6, 9) relative to the substrate. Samples were analyzed by sequential immunoblotting with a anti-FLAG antibody (A) or a pan-Stat5 antibody (B). Mobility of intact Stat5b (p94) and cleaved Stat5b (p80) is indicated in the left margin.