Figure 5.

Mutational analysis of GtgA–p65 interacting residues. A and B, LUMIER-binding assay. His₆–GST-tagged GtgA variants were incubated with the post-nuclear supernatant of 293ET cells expressing p65 fused via its C terminus to the Renilla luciferase. Following elution, immunoblot analysis was performed using an anti-GST antibody (A), and Renilla luciferase activity was measured to calculate the relative fold binding (B). Immunoblot is representative of three independent experiments. Data are presented as the fold change in Renilla luciferase activity relative to His₆–GST–GtgA^E183A and represents the mean ± S.E. of three independent experiments, for which individual data points are indicated. Statistical significance was computed between GtgA^E183A and each GtgA^E183A variant (*, p < 0.05; **, p < 0.01, ordinary one-way ANOVA with post hoc Dunnett's multiple comparisons test). C, 5 μm His₆–SUMO–p65(20–291) was incubated with 0.1 μm of the indicated His₆–GST–GtgA variant for 5 h at 37 °C. The reaction was then quenched by the addition of 2× Laemmli buffer, and proteins were separated and visualized by SDS-PAGE followed by Coomassie Blue staining. Immunoblot analysis using an anti-GST antibody was done to confirm equal amounts of each GST-tagged effector protein. The Coomassie Blue-stained polyacrylamide gel and immunoblots are representative of three independent experiments. D, quantification of His₆–SUMO–p65(20–291) cleavage in C. Data are presented as percentage cleavage relative to control sample and represent the mean ± S.E. of three independent experiments, for which individual data points are indicated. Statistical significances were computed between WT and each GtgA variant (*, p < 0.05; **, p < 0.01, ordinary one-way ANOVA with post hoc Dunnett's multiple comparisons test).