Generation of macFoxp1tg mice and expression of human FOXP1-flag transgene. (A) Construct used for pronuclear injection of C57 oocytes. Restriction enzymes used to release the CD68-FOXP1-flag sequence from the plasmid are indicated. (B) Murine (90 kDa) and human (85 kDa) FOXP1 expression was examined in bone marrow–derived monocytes/macrophages (Mono/Mac), neutrophils (PMN), and lymphocytes isolated from line 41 macFOXP1tg mice. B- and T-cell lymphocyte subpopulations were isolated by flow sorting of splenocytes. Cells were lysed with SDS-PAGE reduced sample buffer, then immunoblotted with an affinity purified anti-FOXP1 polyclonal antibody.12 Mouse Foxp1 and human FOXP1-flag expression during monocyte differentiation. Semiquantitative PCR (C) and corresponding quantitative PCR (D) of mouse Foxp1 and human FOXP1-flag from RNA isolated for peripheral blood monocytes (Mono) and thioglyoclate-elicited macrophages (Mac) isolated at day 3 from wild-type and line 41 and line 57 macFoxp1tg mice. Mean relative expression plus or minus SD; n = 2-3 separate experiments; *P < .04 (P value represents comparison of day 3 macrophage versus monocyte for each group). (E) Murine and human Foxp1 protein expression was examined in bone marrow–derived blood monocytes and thioglycolate-elicited macrophages isolated from macFoxp1tg41 mice at 1 day and 2.5 days. Cells were lysed with SDS-PAGE reduced sample buffer and then immunoblotted sequentially with antiFoxp1 and antitubulin antibodies.
