Fig. 6

The NF-κB is required for TNF-α-induced VCAM-1 expression. a, b Cells were pretreated with various concentrations of Bay11-7082 (NF-κB inhibitor; 0.1, 1, or 3 μM) for 1 h and then incubated with TNF-α for 16 h (a) or 4 h (b). c Time dependence of TNF-α-induced p65 NF-κB phosphorylation, cells were treated with TNF-α (15 ng/ml) for the indicated time intervals in the presence or absence of Bay11-7082 (3 μM). d Cells were transfected with siRNA of p65 or scramble (scrb, as a control) for 24 h and then incubated with TNF-α for 16 h. e Cells were treated with TNF-α for the indicated time intervals in the presence or absence of TNFR nAb (10 μg/ml), c-Src siRNA, or AG1478 (10 μM), LY294002 (10 μM), or Akt siRNA. f Time dependence of TNF-α-stimulated NF-κB transcription activity, cells were transfected with a NFκB-luciferase reporter gene and then exposed to TNFα for the indicated time intervals (upper panel). Moreover, the transfected cells were pretreated with the inhibitor of NF-κB (Bay11-7082), TNFR nAb, c-Src (PP1), EGFR (AG1478), or PI3K (LY294002) for 1 h and then incubated with TNF-α for 6 h (lower panel). The VCAM-1 protein (a, d), mRNA (b), and promoter activity (b, f) were analyzed by Western blotting, real time-qPCR, and promoter assay, respectively. The Akt phosphorylation (c, e) was analyzed by Western blot. Data are expressed as mean ± SEM of three individual experiments (n = 3). ^* P < 0.05, ^# P < 0.01 vs. TNF-α alone