Cross-linking of class II molecules on human dendritic cells induces tyrosine phosphorylation. Monocyte-derived immature DCs (5 × 106) were stimulated at 37°C with medium (lane 1), human LAG-3Ig (lane 2), human IgG1 (lane 3), or I3 (pan class II–specific mAb, lane 4) at 10 μg/mL for 3 minutes followed by cross-linking with a secondary goat antibody directed against human (lanes 2-3) or mouse (lane 4) immunoglobulins at 20 μg/mL for 2 minutes. The cells were lysed, and 75 μg of total cell lysate was loaded per well. Tyrosine phosphorylation of proteins was detected by Western blotting with an antibody specific for phosphotyrosine (anti-pTyr 4G10). The positions of the molecular weight markers are indicated. Lane 1 shows the unstimulated control. Results are representative of 6 experiments performed on different DC samples.
