Fig 4. Macrophage marker expression and responses to TLR2 stimulation are intact in ERK1/2 macrophages.

(A) Bone marrow-derived macrophages grown in LADMAC medium for 7 d were rested overnight in D10F, and then stained for CD11b, CD18, M-CSF receptor and F4/80, and analyzed by flow cytometry. Data are representative of two independent experiments. (B) Bone marrow-derived macrophages from LADMAC-stimulated cultures were rested overnight in D10F, pre-treated for 30 min with U0126 or DMSO vehicle (all others), and then treated with or without 30 nM Pam₃CSK₄ for 15 min, lysed, and analyzed for ERK phosphorylation by Western blotting. Densitometric values (in arbitrary units) for LysM, E1 and ERK1/2, respectively, were 33.12, 30.29 and 36.6 for β-actin; 35.2, 26.5 and 2.94 for ERK2; and 28.88, 34.69 and 6.36 for p-ERK2. Results are representative of two independent experiments. (C) and (D). Bone marrow-derived macrophages were pre-treated with U0126 or DMSO as in (B) and then treated with 30 nM Pam₃CSK₄ for 2 h (C) or 4 h (D). Gene expression was measured by qRT-PCR. These time points were previously determined to demonstrate maximal expression of Il10 and Il12b, respectively [21]. Results represent two independent experiments and show means ± standard deviations. Labels: -, untreated; +, treated with Pam₃CSK₄; B6, C57BL/6J; E1, Erk1 ^-/-; E2, Erk2 ^flox/flox Lyz2 ^Cre/Cre; U, U0126-treated C57BL/6J. ****: P<0.0001, **: P<0.01, *: P<0.05, NS: P>0.05.