Nef reduces surface expression of Fms in 293 cells and increases intracellular gp130Fms, in the presence of Hck. (A) In the left panels, parental 293 cells were transfected with the Fms plasmid, alone or in combination with the plasmid for Nef (CD8-Nef) or Hck (IRES-EGFP), and then stained with PE-labeled anti-Fms. In the right panels, 293 cells stably expressing CD4 were transfected with the indicated plasmids and stained with PE-labeled anti-CD4. These cells were costained with APC-labeled anti-CD8, and the data for cells positive for both CD8 and EGFP are shown. The MFI of the expression of Fms or CD4 is indicated. (B) As in panel A, parental 293 cells were transfected with the Fms plasmid, alone or in combination with the plasmid for Nef or Hck. Total cell lysate was prepared and subjected to Western blotting with antibodies against the N-terminal portion of Fms (N), the C-terminal portion of Fms (C), Hck, Nef, or ERK. (C) 293 cells stably expressing Fms were cotransfected with Nef and Hck (Nef/Hck), or transfected with empty vectors (empty), and then treated with trypsin or control buffer. Total cell lysate was prepared and subjected to Western blotting with antibodies against the C-terminal portion of Fms or ERK. (B,C) The ERK blot is a loading control. The ◀ indicate the position of gp150Fms or gp130Fms.