Figure 4

The role of SIMs and SUMO-1 expression on NF-κB activation by rhTRIM5α. A. 293T cells plated in triplicate were transfected with empty vector (EV), ΔRING/SPRY rhTRIM5α (ΔRS), wild typerhTRIM5α, SIM mutants or RIG-I along with NF-κB-responsive firefly luciferase construct. A renila luciferase construct was used as an internal transfection efficiency control. 48-hours post transfection, cells were lysed and luciferase activity was measured. NF-κB luciferase readings were normalized to renilla luciferase, and plotted as an average fold increase over empty vector. Upper panel, representative luciferase activity in the presence of various constructs.Error bars represent the SEM between the triplicates. Lower panel,representative Western blot. Representative of 4 independent experiments. B. 293A stably expressing wild typerhTRIM5α, K10R mut, SIM mutants with a FLAG-tag or an EV. Cells weretransiently transfected with an NF-κB firefly luciferase and renila luciferase plasmids as in A. Upper panel, NF-κB luciferase activity in the presence of rhTRIM5α mutants. Error bars represent the SD between 3 independent experiments. Lower panel, representative Western blot at the time of data acquisition. C. 293T cells were transfected with Control siRNA or SUMO-1siRNA for 48 hours. Cells were then seeded in a 96-well plate in triplicate and transfected with empty vector or wild type rhTRIM5α. NF-κB activity was measured as in A. Data were plotted by dividing SUMO-1 siRNA activation by control siRNA activation x 100. Inset, representative Western blot. P <0.004 by Student’s t-test. Error bars represent the SEM between the triplicates. Data is representative of 3independent experiments. D. 293T cells were transfected with empty vector, and wild type rhTRIM5α constructs in presence and absence of SUMO-1. NF-κB activity was measured as in A. Data were plotted as in C. Error bars represent the SEM between triplicates. Representative of 3 independent experiments.