Figure 4.

Overexpressed c-IAP2 activates NF-κB. (A) Stimulation of NF-κB-directed transcription. Jurkat cells stably expressing either IκBαWT (filled bars) or IκBαΔN (empty bars) were cotransfected with HIV-κB-CAT (5 μg) and 10 μg of either empty pCMV4 vector or the wild-type c-IAP2 expression plasmid. After 24 hr of growth, the indicated cultures were exposed to TNF (20 ng/ml) for 16 hr. Whole cell extracts were prepared at 40-hr posttransfection and assayed for CAT activity. Results from triplicate transfections are reported as the fold induction in CAT activity (mean ± SEM) relative to that measured in unstimulated Jurkat/IκBαWT cells transfected with HIV-κB-CAT alone (normalized to 1). (B) Induction of NF-κB DNA binding activity. HeLa cells (2 × 10⁶) were cotransfected with pHook-1 (5 μg) and 15 μg of either blank pCMV4 plasmid (lanes 1 and 2) or the c-IAP2WT expression vector (lane 3). Transfected cells were selected by magnetic bead capture (36) after 24 hr of growth. Selected transfectants were propagated for 24 hr and then treated with CHX (50 μg/ml; 4 hr) in the presence (lane 2) or absence (lanes 1 and 3) of TNF (20 ng/ml). Nuclear extracts were prepared and analyzed in gel retardation assays with a radiolabeled κB probe (see Fig. 1A). (C) Stimulation of IκBα degradation. HeLa cells were cotransfected with an expression vector for wild-type IκBα (1 μg) and 10 μg of either pCMV4 (Upper) or c-IAP2WT (Lower). Cytoplasmic extracts were prepared from transfected cells after treatment with CHX (50 μg/ml) for the indicated times. Ectopic forms of IκBα were isolated by immunoprecipitation with anti-FLAG antibodies, resolved by SDS/PAGE, and immunoblotted with IκBα-specific antisera.