Down-regulation of M-CSF receptor is not involved in the impaired proliferation of TF-1-fms-Nef-ER cells induced by Nef activation. (A) Parental TF-1-fms and TF-1-fms-Nef-ER cells were cultured with 4-HT for the indicated periods, and the levels of M-CSF receptor proteins were analyzed by Western blotting. Total cell lysates from TF-1 cells, but not from TF-1-fms cells, in which the M-CSF receptor gene had not been introduced, were included in the analysis as a negative control (fms-negative). (B) TF-1-fms-Nef-ER cells were left untreated (top) or were treated with 4-HT for 24 hours (bottom), and the level of cell surface M-CSF receptor expression was analyzed by flow cytometry with Flag-tagged M-CSF (solid lines). Profiles of cells incubated with a Flag-tagged protein,³⁰  unrelated to M-CSF, are also shown as a control (broken lines).