C1qa^−/− DCs display impaired activation of MAP p38 and ERK1/2 protein kinase after CD40 ligation. GM-CSF DCs from WT and C1qa^−/− mice were incubated alone or with CD40 antibodies (4 μg/mL) or LPS (10 ng/mL). (A) Intracellular staining with antibodies against phosphorylated ERK1/2 (P-ERK1/2) and p38 (P-p38) MAP kinases was evaluated at 10 minutes (LPS) and 30 minutes (CD40Ab) using flow cytometry. The gray histogram represents the phosphorylated form of the protein, whereas the black histogram represents staining with isotype-matched control antibodies. The numbers in the histograms represent the mean fluorescence intensity (MFI) of the antibody to the phosphorylated protein after subtraction of MFI of the isotype control antibody. (B) Phosphorylation of p38 (P-p38) and ERK1/2 (P-ERK1/2) MAP kinases at the indicated time points (0 to 30 minutes) from a different experiment is displayed. Results represent the MFI expressed as arbitrary units (au). Shown are results representative of 2 independent experiments performed with cells from different mice. (C) DCs were stimulated and lysates prepared at the indicated time points. Samples were analyzed by Western blotting using anti–phospho-p38 (P-p38), and anti–phospho-ERK1/2 (P-ERK). Total proteins were evaluated after stripping the membranes and reblotting with anti-p38 (p38), anti-ERK1/2 (ERK), and anti–β-actin (actin) antibodies. The numbers displayed above the bands represent densitometric quantification of the signals.