Fig. 2.
TNF-α induced the translocation of NF-κB p65 and p50 to the nucleus (A) and their nuclear binding to the RE1 and RE2 κB sites in the hα1C1b promoter (B) in primary culture of HCCSMCs. Cotreatment with VIP suppressed both effects of TNF-α. Cells were treated with 20 ng/ml TNF-α for 60 min in the absence or presence of 10−7 M VIP. 10 μg of nuclear extracts were used for each lane. VIP alone did not induce the translocation of NF-κB p65 and p50 to the nucleus or their binding to RE1 and RE2 (NF-κB recognition sites) (data not shown). C and D: TNF-α induced IκBα and IκBβ degradation (C) and IKKα and IKKβ phosphorylation (D) in primary cultures of HCCSMCs. VIP had little effect on the degradation of IκBα , but it blocked the degradation of IκBβ (C). VIP also blocked the phosphorylation of IKKβ and decreased the phosphorylation of IKKα (D). The primary cultures of HCCSMCs were treated with 20 ng/ml TNF-α for various durations in the absence or presence of 10−7 M VIP. Whole cell lyses (10 μg) were used in these experiments. Blots are representatives of 3 independent experiments.