c-Fms/M-CSFR expression in macFoxp1tg peripheral blood monocytes. (A) The level of expression of c-fms was assessed by quantitative PCR using RNA harvested from wild-type and macFoxp1tg blood monocytes. Expression levels were normalized to β-actin and expressed relative to wild-type. (B) Expression of M-CSFR protein in wild-type and macFoxp1tg monocytes was investigated by Western blot analysis of cellular lysates immunoblotted sequentially with anti–M-CSFR and anti-tubulin antibodies. (C) Surface expression of M-CSFR was also investigated by flow cytometry using 2-color staining for monocytes (PE-conjugated MOMA-2) and M-CSFR using FITC enzymatic amplification staining (EAS), as described in Document S1, “Flow cytometry.” Representative flow cytometry for M-CSFR expression on MOMA-2–positive cells isolated from wild-type (WT) and macFoxp1tg mice. M-CSFR–positive cells were quantified by determining percentage gate of the M2 population defined by staining with an isotype-matched control antibody. (D) Overexpression of c-Fms reverses the cytokine production and phagocytosis defects in macFoxptg cells. LPS-stimulated IL-1β and TNF-α production in wild-type and macFoxp1tg bone marrow–dervied monocytes/macrophages retrovirally infected with GFP, GFP.c-Fms, or GFP.kinase-deficient c-Fms (GFP.c-Fms-mu). Values represent mean plus or minus SD (n = 3-5). Phagocytosis was assessed by culturing GFP-expressing cells in the presence of Alexa-conjugated S aureus bioparticles. Cells were viewed for internalization of the particles by fluorescence microscopy after quenching extracellular fluorescence with trypan blue (digital magnification 40×/0.55).