gp130Fms appearing in 293 cells coexpressing Nef/Hck is Golgi-localized underglycosylated Fms. (A) 293 cells stably expressing Fms were transfected with the control CD8 plasmid (empty) or CD8-Nef plasmid (Nef). Similarly, 293 cells stably coexpressing Fms and Hck were transfected with the control CD8 plasmid (Hck) or CD8-Nef plasmid (Nef/Hck). These cells were stained with anti-Fms antibody (top panels). In the bottom panels, 293 cells stably coexpressing Fms and Hck were transfected with CD8-Nef and costained with anti-Fms antibody (green), anti-GM130 antibody (red), and DAPI (blue). (B) 293 cells stably expressing Fms were cotransfected with Nef and Hck (Nef/Hck), or transfected with empty vectors (empty). The total cell lysate was subjected to Fms Western blotting directly (total) or after pull down with WGA-agarose or Con A–agarose. The arrowheads indicate the position of gp150Fms or gp130Fms. (C) Total cell lysate prepared as in panel B was subjected to Fms Western blotting directly (untreated) or after treatment with endo-β-N-acetylglucosaminidase H (Endo H) or peptide-N-glycosidase F (PNGase F).