Figure 5A. Overexpression of MavC but not the inactive mutant MavCC74A in the ∆mavC∆mvcA mutant background causes IκBα accumulation in infected cells. U937 cells infected with the indicated bacterial strains at an MOI of 5 were probed for IκBα. Tubulin was probed as a loading control.B. Prolonged UBE2N modification by MavC restricts bacterial vacuole maturation. HEK293 cells transfected to express the FcγII receptor and 4xFlag-MavC or the C74A mutant were challenged at an MOI of 1 with opsonized L. pneumophila that expresses GFP for 12 h. The vacuole was scored and categorized by the number of bacteria it contained. Data (mean +/- SE) shown were from three independent experiments, each with at least 300 vacuoles counted. *, p<0.05; **, p<0.01; n.s., not significant.C. Overexpression of MavC but not MavCC74A affects intracellular growth of the ∆mavC∆mvcA mutant of L. pneumophila. Raw264.7 cells were infected with the indicated bacterial strains and intracellular bacteria were determined at the indicated time points. Each strain was examined in triplicate and the values shown were mean +/- SE. Similar results were obtained in two independent experiments. *, p<0.1; **, p<0.01; ***, p<0.001(t-test by comparing p(MavC) with p(MavCC74A)).D. MvcA is required for reversal of UBE2N modification during L. pneumophila infection. U937 cells infected with wild type or the ∆mvcA mutant were withdrawn at the indicated time points. Samples resolved by SDS-PAGE were probed for UBE2N, translocated MavC and MvcA. Tubulin was probed as a loading control. Note that in samples infected with the ∆mvcA strain, the level of UBE2N-Ub decreased at slower rates.E. MvcA becomes detectable in intracellular bacteria 5 h after uptake. U937 macrophages infected with wild type L. pneumophila at an MOI of 10 were lysed at the indicated time and recovered bacteria were probed by immunoblotting for MavC and MvcA, respectively. The bacterial isocitrate dehydrogenase (ICDH) was probed sequentially as a loading control.F. MvcA restores the nuclear translocation of p65 inhibited by MavC during L. pneumophila infection. Raw264.7 cells were infected with the indicated L. pneumophila strains for 2 h at an MOI of 1. Fixed cells were immunostained with antibodies specific for p65 and the bacterium, respectively. Host nuclei were labeled with Hoechst. Representative images of cells in each sample category were shown (upper panels); nuclear localization of p65 was determined by scoring at least 300 infected cells (lower panel). Results shown were from three independent experiments done in triplicate and the values shown were mean +/- SE; t-test.G. Overexpression of MvcA but not its inactive mutant MvcAC83A promotes IκBα degradation during L. pneumophila infection. U937 cells were infected with the indicated bacterial strains at an MOI of 5. Lysates of cells infected for the indicated time were probed for IκBα. Tubulin was probed as a loading control. IκBα levels for cells infected with the strains ∆mvA(pMvcA) and ∆mvA(pMvcAC83A) at the indicated time points were evaluated by measuring the band intensity from three independent experiments (lower panel), the values shown were mean +/- SE; t-test.