IL-6–induced protein tyrosine phosphorylation in U266 cells and effect of IFN-β pretreatment. (A) Serum-deprived (14 hours) U266 cells were exposed to IL-6 (25 ng/mL) for the indicated times. Whole-cell extracts equivalent to 8 × 105 cells were analyzed by immunoblotting with an anti-pTyr MoAb (1:1,000 dilution) to detect tyrosine-phosphorylated proteins. Representative of five separate experiments. (B) Serum-deprived U266 cells were treated with various doses of IFN-β (U/mL) for 50 minutes before addition of IL-6 (25 ng/mL) where indicated and subjected to immunoblotting with an anti-pTyr MoAb as in (A). Representative of three separate experiments. (C) U266 cells pretreated with or without IFN-β (500 U/mL) for 50 minutes were induced with IL-6 (25 ng/mL) for 10 minutes. Lysates were analyzed by immunoblotting with a MoAb to pTyr (4G10) followed by stripping and reprobing with a MoAb recognizing Erks. Representative of three separate experiments. Proteins discussed in the text are indicated by arrows. The sizes of molecular mass markers (Prestained SDS-PAGE standards, broad range, and low range; Bio-Rad Laboratories, Richmond, CA) are indicated in thousands of daltons.
