DC-SIGN ligation on MDDCs does not induce maturation but alters the profile of phosphotyrosine-containing proteins. (A) Immature MDDCs were isolated and either not stimulated (NS) or treated with lipopolysaccharide (LPS) or monoclonal antibodies against CD38 (HB136) or DC-SIGN (MR-1) at 20 μg/mL (1X) or 60 μg/mL (3X). After 48 hours, cells were collected and the cell surface expression of CD83 and CD36 was determined by flow cytometry, using an isotype-matched anti–c-Myc antibody (9E10) as control. The percentage of marker-positive cells and the mean fluorescence intensity are indicated in each case. Three experiments were performed on MDDCs from independent donors, and a representative experiment is shown. (B) DC-SIGN ligation induces changes in the pattern of tyrosine phosphorylation in MDDCs. Cells were either left untreated (not stimulated, NS) or incubated with monoclonal antibodies against DC-SIGN (MR-1) as ligation agent, or with MR-1 plus a cross-linking secondary antibody (anti–mouse F(ab′)2, anti–mouse IgG). After 5 minutes, cells were lysed, and the lysates were probed for phosphotyrosine residues by Western blot using the RC20-HRP monoclonal antibody. As a control, a monoclonal antibody against c-Myc (anti–c-Myc) was used. Two experiments on MDDCs from independent donors rendered similar results, and one of them is shown.
