Nef causes tyrosine phosphorylation of Hck, constitutive association of Hck with M-CSF receptor complex, and inhibition in tyrosine phosphorylation of M-CSF receptor in 293T cells. (A-C) 293T cells were transfected with M-CSF receptor (c-fms) expression plasmid, alone or in combination with Nef or Hck expression plasmids. Total cell lysates were prepared and subjected to immunoprecipitation/Western blotting analyses. (C) Transfected 293T cells were treated in the absence or presence of M-CSF for 2 minutes before lysis. (A) Immunoprecipitates with anti-Hck antibody were analyzed with antiphosphotyrosine (pTyr) antibody (top) or anti-M-CSF receptor antibody (middle). The blot shown in the bottom panel, in which the immunoprecipitates were analyzed with anti-Hck antibody, is a loading control experiment for upper panels. (B) Immunoprecipitates with anti-M-CSF receptor antibody were analyzed with anti-Hck antibody (top). The blot shown in the bottom panel, in which the immunoprecipitates were analyzed with anti-M-CSF receptor antibody, is a loading control experiment for the top panel. (C) Immunoprecipitates with anti-M-CSF receptor antibody were analyzed with anti-pTyr antibody (top). The blot shown in the bottom panel, in which the immunoprecipitates were analyzed with anti-M-CSF receptor antibody, is a loading control experiment for the top panel.