Fig. 5

Vpx blocks p65 nuclear translocation. A Immunoblot from HEK293T cells that had been transfected for 24 h with 2 µg FLAG-tagged SIVmac Vpx, vaccinia virus protein B14 or EV control and stimulated for 0, 15 or 30 min with 50 ng/ml TNFα. Blots were probed with antibodies against total p65, phosphorylation of p65 on serine 536 (pp65), total IκBα, phosphorylated IκBα (pIκBα), FLAG for Vpx and B14 expression and actin. Signal intensity for total IκBα normalised to actin and pp65 normalised to total p65 are shown underneath. B NF-κB reporter activity from HEK293T cells transfected in parallel with the experiment from A and stimulated with 50 ng/ml TNFα for 8 h. Statistical analyses were performed using Student’s t‐test, with Welch’s correction where appropriate. ***P < 0.001. C Single cell analysis quantifying the Integrated Nuclear Intensity of NF-κB p65 in HeLa cells transfected with FLAG-tagged SIVmac Vpx or EV control and stimulated for 0 and 30 min with 50 ng/ml TNFα. Horizontal lines indicate the mean. Kruskall–Wallis test with Dunn’s multiple comparison, ****P < 0.0001. D Representative example of immunofluorescence staining of NF-κB p65 (green) after FLAG-tagged SIVmac Vpx or EV control transfection and stimulated for 0 and 30 min with 50 ng/ml TNFα. FLAG-tagged Vpx (magenta). Nuclei are outlined in white. Data in 5A are from a representative experiment repeated 3 times. Data in 5B are mean ± SD, n = 3, representative of 3 repeats