Figure 2.
Effect of WT and mutant SSI-1 on IL-6 signaling. (A) COS7 cells were cotransfected with the reporter gene for the IRF-1 promoter and pEF-BOS carrying either WT, mutant SSI-1, or the vector alone. Luciferase activity (AU, arbitrary units) was measured after stimulation without (−) or with (+) IL-6 (100 ng/ml) plus soluble IL-6 receptor (50 ng/ml). Each bar represents the mean ± SD of three experiments. Levels of expression of the SSI-1 mutants were visualized by blotting cell lysates with anti-SSI-1 mAbs. FL238 recognizes the SH2 domain of SSI-1, and 1262B recognizes an N-terminal portion of SSI-1. (B) M1 cells stably transfected with cDNA for WT, Δ49, R/Q, ΔSC, or Neo gene alone were seeded with or without IL-6 (100 ng/ml) plus soluble IL-6R (50 ng/ml) at day 0. The numbers of viable cells were determined by trypan blue exclusion assay and counted in a hemocytometer on days 1–4. Averages of three independent clones of M1/SSI-1 transfectants are shown. Vertical bars indicate SD.