Activation of IKK activity by GMCSF. (A) HUVECs were treated with GMCSF for the indicated time periods, followed by immunoprecipitation of endogenous IκB kinases with anti-IKK agarose. In vitro kinase assays were done with recombinant wild-type GST-IκBα or mutant IκBα (with Ser32 and Ser36 mutated to alanine) as described in “Materials and methods.” Radioactively labeled IκBα was resolved by SDS-PAGE, detected with PhosphorImager equipment, and quantified as indicated in the lower panel. (B) Degradation of IκBα: HUVECs or TF-1 cells (GMCSF-starved for 24 hours) were treated with GMCSF as indicated, followed by preparation of cell extracts, SDS-PAGE, and immunoblotting for detection of endogenous IκBα.