DC-SIGN ligation results in ERK and PLC-γ activation in transfected Jurkat cells and promotes transient calcium mobilization in MDDCs. (A) DC-SIGN on Jurkat-DC-SIGN transfectants was ligated by the anti–DC-SIGN MR-1 antibody, and the cells were incubated at 37°C for 5 minutes. As a control, cells were incubated with either anti-CD38 or anti-CD3 monoclonal antibodies. Mock-transfected cells were subjected to the same treatments for control purposes. After cell lysis, phosphorylated ERK (pERK), p38 (pp38), and PLC-γ (pPLC-γ), and total levels of ERK and PLC-γ were detected using specific polyclonal antisera. The experiment was performed twice with similar results, and one of the experiments is shown. NS indicates not stimulated. (B-C) Calcium determination in MDDCs after DC-SIGN ligation. Fluo-3AM–loaded MDDCs were left untreated (B) or treated with 50 nM SDF-1α (C), and subsequently incubated with anti–DC-SIGN MR-1 monoclonal antibody (20 μg/mL) (right panel) or an isotype-matched control antibody (left panel). Calcium flux was determined by flow cytometry at the indicated time points. Arrows indicate the time of addition of the MR-1 monoclonal antibody. Similar results were obtained from 3 independent experiments, and 1 of them is shown.