Analysis of MAPK and NK-κB activation and surface molecule expression (CD83, RAGE) in G-β₂GPI-stimulated DCs. (A) p38 MAPK and ERK activation in DCs stimulated with G-β₂GPI. iDCs (5 × 10⁴ cells) stimulated for 30 minutes with G-β₂GPI (10 μg/mL), glucose (5mM), LPS (0.2 μg/mL), or PMA (0.2 μg/mL) were analyzed by cell-based ELISA MAPK assay to monitor p38 and ERK activation. The number of cells in each well was counted and normalized using crystal violet solution, and the results are expressed as arbitrary units. G-β₂GPI induced the activation of both the p38 MAPK and the ERK pathways (n = 6, P = .001). (B, C) Flow cytometric analysis of CD83 and RAGE expression in DCs stimulated with G-β₂GPI after pretreatment with specific inhibitors for the MAPK family. The increase in CD83 and RAGE expression after G-β₂GPI stimulation was significantly prevented by pretreatment of iDCs with the p38 MAPK inhibitor SB203580, whereas the pretreatment of iDCs with the ERK inhibitor PD98059 prevented only the up-regulation of RAGE expression (n = 3, CD83: *P < .01 and ^†P < .05 comparing G-β₂GPI vs G-β₂GPI + SB; RAGE: *P < .001 and ^†P < .001 comparing G-β₂GPI vs G-β₂GPI + SB and ^‡P < .001 comparing G-β₂GPI vs G-β₂GPI + PD). (D) NF-κB activation in G- and M-β₂GPI–stimulated DCs. In G-β₂GPI- and M-β₂GPI–stimulated DCs, active p65 and p50 levels were significantly increased compared with unstimulated iDCs. Pretreatment of iDCs with saturating concentrations of the blocking anti-RAGE mAb prevented the up-regulation of active p65 in response to G-β₂GPI, but not in response to M-β₂GPI (n = 6, p65: *P ≤ .001, ^†P < .01, and ^‡P < .001 comparing G-β₂GPI vs M-β₂GPI and ^§P = .001 comparing G-β₂GPI vs α-RAGE + G-β₂GPI; for p50: *P ≤ .001).