Oscillations in AKT and ERK1/2 phosphorylation require CSF-1R. (A) Monocytes were treated for indicated times with 100 ng/mL CSF-1. (Top) Flow cytometric analysis of CSF-1R expression at the surface of monocytes exposed for indicated times to 100 ng/mL CSF-1 (mean fluorescence indexes [MFIs] normalized to untreated cells). (Bottom) Immunoblot analysis of CSF-1R expression in total cell lysates. HSC70 indicates loading control. One representative of 3 independent experiments is shown. (B) Two examples of flow cytometric analyses are shown, before (0) and after 8 hours of CSF-1 treatment (8h). Isotype-matched control: empty histogram. (C) Monocytes were cultured for indicated times in the presence of 100 ng/mL CSF-1 alone or with 1 μmol/L GW2580 (GW) added either 30 minutes before CSF-1 treatment (P) or 30 minutes, 2 hours, or 6 hours after CSF-1. Immunoblot analysis of indicated proteins is shown. Two exposures of pCSF-1R are shown. Molecular weight (MW) is given in kilodaltons. HSC70 indicates loading control. (D) Monocytes were treated as shown previously and CSF-1 was kept (left) or removed from the culture medium (washing) at 2 hours before immunoblot analysis of indicated proteins.