FIG. 4.
Activation of IKKs by A224L. (A) Cleared extracts from 106 pcDNA-A224L stably transfected Jurkat cells were incubated and immunoprecipitated with 1 μl of NEMO antiserum or with anti-c-Rel antibody as a control. Immunoprecipitates were separated by SDS-PAGE and revealed with a monoclonal antibody against IKKs. (B and C) Cleared extracts from pcDNA- and A224L-transfected cells were immunoprecipitated with 1 μl of NEMO antiserum and used in an in vitro kinase assay as described in Materials and Methods, using (B) 0.5 μg of GST(1-115)-IκB-α or (C) 0.5 μg of mutated IκB-α as the substrate. Proteins were separated by SDS-10% PAGE and developed by autoradiography. Data were reproducible for at least three separate kinase assays performed under the same conditions.