Nonpersistent ERK1/2 phosphorylation in CD1c⁺PBDCs and migratory MoDCs activated with CD40L₃plus IFN-γ. CD1c⁺ PBDCs were isolated from PBMCs. Following a 24-hour culture in the presence of GM-CSF plus IL-4, CD1c⁺ PBDCs (5 × 105 per condition) were stimulated with CD40L₃ plus IFN-γ for 30 minutes or 24 hours. Subsequently, CD1c⁺ PBDCs were harvested, washed, resuspended at concentrations of 5 × 106/mL in Western sample buffer, and snap frozen. ERK1/2 phosphorylation of 1 representative experiment is shown (n = 3 for CD1c⁺ PBDCs). Immature MoDCs were stimulated with CD40L₃ plus IFN-γ directly following the 7-day culture period (n = 5). Migratory MoDCs were initially stimulated with GM-CSF plus TNF-α plus IFN-α plus PGE₂ for 24 hours and then washed, and CD40L₃ plus IFN-γ was added as indicated (n = 4).