Figure 6.

SEA induced processing occurs in a p38-dependent manner. A. Extracts from DCs pulsed for 10min, 30min, 1hr or 2hr with SEA or LPS were electrophoretically separated, blotted, probed with Ab specific for phosphorylated p38 and to serve as a loading control, reprobed with Ab specific for unphosphorylated p38. B. In order to determine if p38 signaling was required for SEA-mediated antigen-processing, DCs were pulsed with HRP for 1hr, washed and cultured in the presence of p38 specific inhibitors SB203580, SB202190 or PD169316 for 1hr prior to activation with SEA. After 19hrs, DCs were harvested as described earlier for assessment of antigen processing capacity. C. In order control for effects of inhibitors on basal antigen -processing levels, the ratio between the percentage of active HRP remaining in SEA-activated and immature-DCs was calculated for each inhibitor treated group. Significant differences by Student's t test (** p<0.01), where data points represent mean value ± SD of data from three experiments. D. The effects of p38 inhibition on the ability of SEA to induce antigen processing and presentation by DCs was assessed by inhibiting Ova-pulsed DCs with the p38-specific inhibitor PD 169316 for 1hr prior to overnight stimulation with SEA or TLR ligands, LPS or CpG. DCs were then co-cultured with CFSE labeled naïve CD4⁺ purified DO.11.10 T cells. After 3 days, cells were harvested by assessed for proliferation by CFSE. Data are representative of 2 individual experiments.