NOSTRIN suppresses NFκB- and AKT-signaling pathway and reverses TNFα-induced NFκB activation by directly interacting with TRAF6.
A, Western blotting analysis of proteins from endothelial cells of the four treatment groups evaluating the levels of NFκB1 (p105/p50) and NFκB2 (p100/p52). B, densitometric quantification of proteins from the Western blotting analysis depicted in A normalized to loading control rpL7 using ImageJ software. Error bars represent S.E. from three biological replicates. C, immunoblot analysis of p50 from endothelial cells in the absence or presence of TNF-α (25 ng/ml) stimulation for 15 min (lanes 1 and 2) and of p50 from NOSTRIN-overexpressing endothelial cells in the absence or presence of TNF-α (25 ng/ml) stimulation for 15 min (lanes 3 and 4). D, quantification of p50 levels from C by densitometric analysis normalized to loading control rpL7 using ImageJ software. Error bars represent S.E. from three experiments using three different biological samples. E, immunoprecipitation with anti-TRAF6 antibody followed by immunoblotting (WB) using anti-NOSTRIN antibody in endothelial cells transfected with empty vector or NOSTRIN cDNA. The experiment was repeated three times to ensure reproducibility. F, Western blotting analysis of p-AKT and AKT from endothelial cells from the four treatment groups. RpL7 was used as a loading control. G, densitometric quantification of p-AKT protein level relative to total AKT protein from three replicates using ImageJ software. Error bars represent S.E. from three biological replicates. *, p < 0.05; **, p < 0.01; and ***, p < 0.005.