Staurosporine prevents LLnL-induced stabilization of STAT5 and βc phosphorylation. IL-3–depleted Ba/F₃ cells were treated with LLnL for 3 hours then stimulated with IL-3 for 0 to 2 hours. (A) After 30 and 60 minutes stimulation, staurosporine and vanadate were added as indicated and the incubation continued for a further 60 minutes. Nuclear extracts were prepared and the binding to³²P-labeled oligonucleotide STAT5 probe determined. (B) Nuclear extracts from A (25 μg) were separated by SDS-PAGE (6% gel), transferred, and immunoblotted with antiphosphotyrosine-STAT5 antibody. (C) In a separate experiment, after 30 and 60 minutes stimulation, staurosporine was added and the incubation continued for a further 60 minutes. βc immune complexes (10⁷ cells) were separated by SDS-PAGE (7% gel), transferred, and immunoblotted with RC20 antibody. Whole-cell extracts were also prepared by lysis in 0.1% SDS and 50 μg samples were separated by SDS-PAGE (6% gel), transferred, and immunoblotted with anti-STAT5b antibody. In panels A through C, the position of relevant bands are indicated.