FIG. 4.

(A and B) Inducible expression of dn STATs and dn ras. Each clone was treated with 0.5 mM IPTG for the times indicated. HA-immunoprecipitated proteins (A) or total cell lysates (B) were subjected to SDS-PAGE. The blots were probed with anti-STAT1, anti-STAT3, anti-STAT5b, and anti-ras Abs. (C) Effects of dn mutants on each signaling pathway. Four types of reporter plasmids, each containing 3× ISRE, 4× APRE, 3× β-Cas, and 3× AP1, were used as reporter genes. F-36P-mpl cells were electroporated with 30 μg of the reporter gene together with 30 μg of pRL-CMV-Rluc. The cells were serum and IL-3 starved for 12 h and then stimulated with rhTPO (30 ng/ml) or rhIL-3 (100 ng/ml) for 5 h. To examine the effects of dn mutants, the cells were pretreated with 0.5 mM IPTG for 24 h before electroporation and cultured with IPTG during the assay. Relative firefly luciferase activities were calculated by normalizing transfection efficiency to renilla luciferase activities. The results shown are means ± the standard deviations of triplicate experiments. Open bars, unstimulated.