FIG 5.

CUL4A and CUL4B are both coisolated with Vpr and Vpx. Cultures of HEK293T cells were transfected with expression vectors for untagged HIV-1 Vpr or FLAG-epitope-tagged HIV-1 Vpr, SIV Vpr, or SIV Vpx. Twenty-four hours after transfection, the cells were lysed, and insoluble debris was removed by centrifugation. Cleared lysates were incubated with FLAG-specific-antibody-coated beads to isolate the epitope-tagged viral proteins. The isolated viral proteins were subsequently eluted from the beads by competition with FLAG peptide. Proteins present in the eluates were resolved by SDS-PAGE and identified by Western blotting with specific antibodies. Preimmunoprecipitation (pre-IP) samples were derived from the lysates prior to incubation with antibody-coated beads. Untagged HIV-1 Vpr served as a control to ensure that the identified proteins were isolated through interactions with tagged viral proteins rather than nonspecific interactions with the antibody-coated beads.