Evaluation Stat5 protease activity in mutant FdCP1 cell lines.

(A) Recombinant Stat5b (rStat5b) was incubated for 4 hours at 37°C with either 35 μg (lanes 1, 8, 11, 15, 18) or 70 μg (lanes 2, 9, 12, 16, 19) CHAPS extract from Ba/F3 (lanes 1-3), FdTrk (lanes 4-6), 19-neo (lanes 8-10), 19-WT (lanes 11-13), 19-M1 (lanes 15-17), and 19-M2 (lanes 18-20) cells. Protease activity was blocked in some samples by the addition of 0.5 mmol/L phenylmethylsulfonyl fluoride (+). Control samples of undigested rStat5b are found in lanes 7 and 14. Digestion was evaluated by immunoblotting with pan-Stat5 antibody. Mobility of intact endogenous Stat5a (p96), rStat5b (p94) and cleaved Stat5b (p80) is indicated in the left margin. (B) Equivalent amounts of CHAPS extracts are evaluated for contamination with Stat5b by immunoblotting with pan-Stat5 antibody. Mobility of intact endogenous Stat5a (p96), Stat5b (p94), and truncated Stat5b (p80) is indicated in the left margin.