FIG. 2.
IKKβ phosphorylates A20 on serine 381 in vitro. (A) Flag-A20 was transfected into HEK293T cells and immunoprecipitated using an anti-Flag antibody. Immunoprecipitates were incubated with purified GST-IKKβ in the presence of BMS-345541 (3 μM) or vehicle control for 20 min at room temperature. [γ-32P]ATP was then added to the kinase reaction mix, and an in vitro kinase assay was performed. (B) IKKβ was cotransfected into HEK293T cells with Flag-A20. Flag-A20 was stringently immunopurified and subjected to SDS-polyacrylamide gel electrophoresis. Coomassie staining identified the purified Flag-A20, which was then excised from the gel and submitted for analysis by LC/MS/MS. (C) LC/MS/MS identified phosphorylation of A20 at serine 381.