In human cells, SIV
Vpr does associates less efficiently with the CRL4 E3 ubiquitin ligase. (A) 293 T cells were cotransfected with HA-Vpr, HA-VprR90K, or SIVagm HA-Vpr and Flag-DCAF1 expression vectors. Vpr was immunoprecipitated with anti-HA antibody and the immunoprecipitates were analyzed on an immunoblot probed with anti-Flag MAb, anti-DDB1 antibody, and anti-HA antibody. (B) HeLa cells were transfected with HA-Vpr or SIVagm HA-Vpr expression vector. The transfected cells were permeabilized, fixed, and then incubated with anti-DDB1 and anti-HA antibodies followed by Alexa Fluor 594-anti-rabbit IgG and Alexa Fluor 488-anti-rat IgG. Representative images from an HA-Vpr or SIVagm HA-Vpr expressing sample are shown. Arrows indicate Vpr foci and DDB1 dots which colocalize. (C) The percentage of Vpr foci colocalized with DDB1 among total Vpr foci was calculated. More than 170 Vpr foci were evaluated for each sample and three independent experiments were done. The data are the mean values with standard deviations. P values were calculated by the Student’s t-test with P < 0.05 considered significant.