Inhibition of IL-4-induced STAT6 activation by treatment of CLL cells with PF-956980. (A-C) CLL cells were incubated for 20 minutes with the indicated concentrations of IL-4. When present, 1μM PF-956980 (abbreviated as PF) was added to cultures 20 minutes before the addition of IL-4. Protein lysates were analyzed by Western blotting using the antibodies indicated. In this and all figures, the densitometric band intensities determined using phosphorylation state-specific antibodies were normalized with respect to the intensities obtained by application of the corresponding phosphorylation state-independent antibody to a duplicate blot. The normalized band intensities are shown numerically. (D) CLL cells were incubated in the presence of the pan-caspase inhitor, Z-VAD-FMK (100μM), and with additions of IL-4 and/or PF-956980, as indicated. Lysates were prepared for Western blot analysis after 18-hour incubation. The isolates used here were from patients CLL1 (A), CLL 12 (B), CLL 19 (C), and CLL 28 (D). The data in each panel are representative of experiments carried out using isolates from 4 to 7 patients.