PG490 inhibited both IKKα and IKKβ kinase activity. T cells were pretreated with various concentrations of PG490 for 2 h and then stimulated with TNF-α for 10 min (A), PMA + ionomycin for 15 min (B) or CD3/CD28 for 30 min (C). Cell pellets were collected and total cell lysates were prepared and analyzed for the kinase activity of IKKα and IKKβ by immunoprecipitation kinase assays. The total IKKα and/or total IKKβ levels were determined by Western blotting. The analysis of PG490-mediated suppression of IKKα and IKKβ activities induced by TNF-α stimulation was performed on pooled data from T cells from 3 different donors (D). *, P value of <0.05.