Ligation of hDCAL-2 on iDCs induces activation of kinases and receptor internalization. iDCs were stimulated with anti-DCAL-2 mAb for the indicated time periods before being lysed. Total cell lysates were used for Western blot and probed with (A) antiphosphorylated tyrosine Ab (4G10), (B) anti-phospho-p38 MAPK, anti-phospho-ERK antibody, and anti-total p38MAPK, and anti-ERK as loading control. (C) iDCs were stimulated with anti-DCAL-2 mAb or IgM control for 15 minutes. After washing with cold PBS, cells were fixed and then permeabilized in 90% methanol. Cells were then washed twice and stained with anti-phospho-ERK1/2-PE and anti-phospho-p38 MAPK-Alexa fluor 488. The cells were analyzed by FACS. (D) The internalization of DCAL-2 after ligand binding was measured after coating iDCs with anti-DCAL-2 using GAM Ig-FITC and flow cytometry as described in “Materials and methods.” Results are representative of 3 independent experiments.