Ubiquitin conjugating enzyme variant (Uev)1A activates NF-κB in MDA-MB-231 cells in a Ubc13-dependent manner. (A) NF-κB activation in UEV-overexpressing cells. Nuclear or whole-cell extracts were prepared, equal amounts of protein were separated by SDS–PAGE gel, followed by western blotting analysis using an anti-p65 antibody to measure NF-кB nuclear enrichment and an anti-P(S32)-inhibitor of NF-κBα (IκBα) antibody to assess the degree of IκBα phosphorylation as an indication of its degradation and release of NF-κB into the nucleus. (B) NF-κB activation in Uev1-depleted cells. Experimental conditions are as described in Figure 4A. (C) The F38 residue of Uev1A was required for its interaction with Ubc13. MDA-MB-231 cell extracts expressing UEV1A or UEV1A-F38E were immunoprecipitated with an anti-HA monoclonal antibody, followed by western blotting with an anti-Ubc13 monoclonal antibody and an anti-HA monoclonal antibody. (D) Uev1A-F38E failed to activate NF-κB. Experimental conditions were as described in Figure 4A.