Nef activation causes perturbations in M-CSF receptor signaling in TF-1-fms-Nef-ER cells. (A-C) TF-1-fms-Nef-ER cells were deprived of M-CSF for 14 hours and restimulated with M-CSF for the indicated periods. For the activation of Nef, 4-HT was added to the culture at the initiation of M-CSF deprivation. Total cell lysates or immunoprecipitates were analyzed by Western blotting. (A) Total cell lysates from untreated or 4-HT-treated cells were analyzed with antiphosphotyrosine (pTyr) antibody. See “Mechanism by which Nef inhibits M-CSF receptor signaling” for explanations of the symbols in detail. (B) Immunoprecipitates with anti-pTyr antibody were analyzed with anti-ERK antibody (top). Alternatively, total cell lysates were analyzed with antibody specific for phosphorylated ERK (middle). The blot shown in the bottom panel, in which the total cell lysates were analyzed with -ERK antibody, verified that comparable amounts of proteins were loaded in the top panel. (C) Immunoprecipitates with anti-pTyr antibody were analyzed with anti-M-CSF receptor (c-fms) antibody (top). Alternatively, the immunoprecipitates with anti-M-CSF receptor antibody were analyzed with anti-pTyr antibody (middle). The blot was exposed to autoradiography film for 10 seconds (short) or 1 minute (long). See “Mechanism by which Nef inhibits M-CSF receptor signaling” for explanations of the symbols in detail. 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 upper panel. (D) TF-1-fms-Nef-ER cells were cultured with or without 4-HT for 14 hours under M-CSF-free conditions. Total cell lysates were prepared and subjected to immunoprecipitation with anti-M-CSF receptor (c-fms) antibody or anti-Hck antibody. Anti-M-CSF receptor immunoprecipitates were analyzed with anti-Hck antibody or anti-M-CSF receptor antibody. Anti-Hck immunoprecipitates were analyzed with anti-pTyr antibody, anti-M-CSF receptor antibody, or anti-Hck antibody.