IFN-β pretreatment attenuates IL-6–induced tyrosine phosphorylation of Jak2. (A) Whole-cell extracts were prepared from serum-deprived (14 hours) U266 cells pretreated with IFN-β (500 U/mL) for 50 minutes and then exposed to IL-6 (25 ng/mL) for the times indicated. Jak2, Jak1, and Tyk2 were sequentially immunoprecipitated from the same extracts and subjected to immunoblotting with an anti-pTyr MoAb. (B) The blot shown for Jak2 in (A) was stripped and reprobed with the immunoprecipitating MoAb to Jak2 to demonstrate equal loading and that the higher molecular mass bands detected with the anti-pTyr MoAb were not cross-reacting proteins recognized by the anti-Jak2 MoAb. The Jak/Tyk-specific bands are denoted by arrows. Representative of three separate experiments.