Fig. 3

Vpx-mediated inhibition of NF-κB is independent of DCAF1, SAMHD1 and HUSH. A NF-κB reporter activity from HEK293T cells co-transfected for 24 h with 50 ng SIVmac WT, E15AE16A SAMHD1 mutant or Q47AV48A HUSH mutant Vpx or EV control plus 0, 1.25, 2.5 or 5 ng each of FLAG-cGAS and FLAG-STING per well. B NF-κB reporter activity from HEK293T cells co-transfected for 24 h with 50 ng SIVmac WT, E15AE16A SAMHD1 mutant or Q47AV48A HUSH mutant Vpx or EV control plus 25 ng p65 per well. Immunoblot detecting Vpx mutants using an antibody against the FLAG tag and actin. C NF-κB reporter activity from HEK293T cells co-transfected for 24 h with 12.5, 25, 50 or 100 ng SIVmac WT or R51AS52A STING mutant Vpx or EV control plus 25 ng p65 per well. D NF-κB reporter activity from HEK293T cells co-transfected for 24 h with 12.5, 25, 50 or 100 ng SIVmac WT or Q76R DCAF1 mutant Vpx or EV control plus 25 ng p65 per well. E Immunoblot from NF-κB reporter activity in HEK293T cells from C, D detecting Vpx mutants using an antibody against the FLAG tag and actin. The highest transfection dose was selected. F NF-κB reporter activity from HEK293T cells previously transfected for 48 h with siRNA against DCAF1 (siDCAF1) or Ctrl siRNA (siCtrl) and then co-transfected for 24 h with 50 ng SIVmac or EV control plus 25 ng p65 per well. G Normalised NF-κB reporter activity from F. H Immunoblot for DCAF1 depletion in NF-κB reporter activity in HEK293T cells from F, G detecting DCAF1, actin and Vpx using a Vpx antibody. Data are mean ± SD, n = 3, representative of at least 3 repeats. Statistical analyses were performed using Student’s t‐test, with Welch’s correction where appropriate. *P < 0.05, **P < 0.01, ***P < 0.001