Kinetics and strength of activation of ERK1/2, p38 MAPK, and NF-κB in MoDCs in response to different CD40L preparations. MoDCs were stimulated as described in the legend to Figure 1. Cells were harvested at the indicated time points following stimulation, gently washed, and resuspended in lysis buffer. (A) Phosphorylation of ERK1/2 and p38 MAPK 30 minutes and 48 hours after activation (representative of at least 4 experiments). (B) NF-κB binding activity in cellular protein extracts of migratory-type and proinflammatory-type MoDCs. Electrophoretic mobility shift assay (EMSA) was performed 1 hour (n = 1) and 48 hours (n = 3) after stimulation. Supershift: comparison of binding activity of NF-κB family members in CD40L₁- and CD40L₃-activated MoDCs after 48 hours. (C) NF-κB binding activity as percentage reduction of optical density in the main band in the presence of the specified antibody relative to the control band without antibody. Results are shown as mean values ± SEM of 3 experiments; *P < .05 comparing CD40L₁ and CD40L₃.