Nef induces Golgi localization of active Hck. (A) Parental 293 cells were transfected with the control CD8 plasmid or CD8-Nef plasmid, and then stained with anti-CD8 antibody (green), anti-GM130 antibody (red), or DAPI (blue). (B) In the top panels, parental 293 cells were transfected with wild-type Hck, or cotransfected with wild-type Hck and Nef, and then stained with the antibody specific for active Hck (ie, Hck phosphorylated at Tyr411). In the middle panels, parental 293 cells cotransfected with wild-type Hck and Nef were costained with anti-Hck pTyr411 antibody (green), anti-GM130 antibody (red), and DAPI (blue). In the bottom panels, parental 293 cells were transfected with the constitutive-active AxxA Hck (see Figure 4B), or cotransfected with the AxxA Hck and wild-type Nef, and then stained with anti-Hck pTyr411 antibody. See “Western blotting, flow cytometry, and immunofluorescence with 293 cells” for image acquisition information.