Activation of Hck by Nef is essential but not sufficient for accumulation of gp130Fms. (A) The Nef mutants used (M20A, WL/AA, AxxA, and LL/AA) are schematically shown. All the constructs are CD8-Nef chimeras. 293 cells stably expressing Fms were cotransfected with wild-type Hck and the plasmid indicated, and then analyzed for the expression of Fms, Nef, Hck, or ERK by Western blotting. (B) Schematic representations of Hck and the mutants used. KE is the kinase-dead form, whereas AxxA and YF are the constitutive-active forms.¹⁴  293 cells stably expressing Fms were transfected with empty vectors (empty), Nef plasmid (Nef), or the indicated Hck plasmid (“w/o Nef” lanes), or cotransfected with wild-type Nef and the indicated Hck plasmid (“with Nef” lanes). Then, the transfected cells were analyzed for the expression of Fms, Nef, Hck, or ERK by Western blotting. (A,B) The ERK blot is a loading control. The ◀ indicate the position of gp150Fms or gp130Fms.