Figure 2

RelB sub-cellular localization and alternative NF-κB activity in 22Rv1-derived cells. A) Cellular distribution of the main NF-κB subunits analyzed by immunoblotting on protein extracts of cytoplasmic and nuclear compartments from RelB clones and GFP control cells. Actin was used as loading control for both cytoplasmic and nuclear protein extracts whereas GAPDH was used as a purity indicator for nuclear protein extracts. B) Immunoprecipitation of RelB from cytoplasmic and nuclear compartments protein extracts from RelB clones and GFP control cells. Immunoblot analyses of p100, p52 and p65 were performed from immunoprecipitated RelB fraction. C) NF-κB transcriptional activity by luciferase gene-reporter assay. RelB clones and GFP control cells were co-transfected with p3enh-κB-CONAluc (Firefly luciferase) and phRL-CMV (Renilla luciferase). Normalized data for each cell population presented are the luminescence ratio Firefly luc/Renilla luc. Experiments were done three times in triplicate. Error bars represent the standard error of the mean and p < 0.05 was considered a significant variation (Mann- Whitney U test).