Figure 5.

The two NFκB signaling pathways are of varying cell type-specific relevance for TWEAK-induced TRAF1 expression. (A) Cells were stimulated overnight with Flag-TWEAK (200 ng/ml) and Flag-TNF (100 ng/ml) and total cell lysates were analyzed by western blotting for NIK accumulation, p100 processing and expression of TRAF1, where indicated cells were pretreated for 30 min with 10 μM TPCA1. (B) Control vector and IκBα-SR transduced A172 cells were stimulated overnight with increasing concentrations of Flag-TWEAK and were assayed by western blotting of total cell extracts for TRAF1 production and activation of the alternative NFκB pathway (left panel). Efficacy of IκBα-SR-mediated repression of the classical NFκB pathway was proved by analyzing phosphorylation and degradation of IκBα in cells stimulated with TNF (100 ng/ml) (right panel). (C) The indicated cell lines were pretreated or not with 10 μM MLN4924 for 30 min and then stimulated with Flag-TWEAK (200 ng/ml) and Flag-TNF (100 ng/ml). The next day, total cell lysates were prepared and analyzed for the presence of the indicated proteins by western blotting.