FIGURE 7.

Nef interacted with Gα_i2in vitro and induced Gα_i2 ubiquitination. A, varying amounts of purified GST or GST Nef were incubated with cellular extracts of Jurkat cells or human monocytes. Bound proteins were resolved by SDS-PAGE, and Gα_i species were detected by immunoblotting. B, Nef-induced ubiquitination of native Gα_i2 in Nef-expressing CEM cells. CEM cells were co-transfected with FLAG-tagged ubiquitin and Nef or null plasmid. Cellular extracts were directly immunoblotted for Gα_i2 (B1 and B2, left panels) or immunoprecipitated first with anti-FLAG polyclonal antibody (B1) or mAb (B2) followed by immunoblotting for Gα_i2. Asterisks denote Gα_i2 (left) and mono- and diubiquitinated Gα_i2 (right). Numbers (kDa) refer to molecular mass markers. Data are representative of three experiments. C, Nef-mediated Gα_i2 breakdown was partially rectified by Dynasore, a small molecular weight inhibitor of dynamin, or the proteosome inhibitor epoxomycin. Cellular extracts were analyzed by immunoblotting for Gα_i2. Gα_i2 bands are shown pairwise for Nef(+) versus Nef(−) for each treatment with relative (%) density (average values from three experiments) denoted below.