Figure 3

PG27 prevented IκBα degradation and reduced the activity of IKKα but not IKKβ. In (A), T cells were pretreated with PG27 (200 ng/mL) for 2 h or left untreated, and then stimulated with TNF-α for various lengths of time as indicated. After washing, cell pellets were collected and cytoplasmic and nuclear extracts were prepared and analyzed for the protein levels of IκBα by Western blotting (upper panel) and NF-κB DNA-binding activity by EMSA (lower panel), respectively. In (B), T cells were pretreated with PG27 for 2 h and then stimulated with TNF-α for 10 min. After washing, cell pellets were collected and total cell lysates were prepared and analyzed for the kinase activity of IKKα (upper panel) or IKKβ (middle panel) by immunoprecipitation kinase assays. The total cell lysates were also analyzed for IKKα protein levels by Western blotting (lower panel). In (C), after pretreatment with PG27, T cells were stimulated with PMA + ionomycin for 15 min, and the cell lysates were collected for measurement of IKKα and IKKβ activity. The densitometric intensities of the individual bands are shown. The analysis of PG27-mediated suppression of TNF-α-stimulated IKKα and IKKβ was performed in T cells from at least 3 different donors (D). *, P value of <0.05.