Comparison of LPS-induced ERK activation and effects of ERK inhibitor on LPS-induced changes in phenotype, chemokine receptor expression, and chemotaxis between CB and AB DCs. (A) LPS induced p-ERK in CB but not AB DCs. One representative of Western blotting data was shown. CB and AB DCs were either not treated (as a control) or treated side by side for 15 minutes and 30 minutes with LPS. Cell lysate was subjected to Western blotting using Abs specific for p-ERK. PVDF membranes were stripped and reprobed with Abs for total ERK as a loading control. (B) Average density of p-ERK band of CB and AB DCs plus or minus SEM from 3 independent experiments as shown in panel A was presented. Density of individual p-ERK bands was calculated relative to that of unstimulated cells (defined as 1.0). (C) PD98059 significantly enhanced LPS-induced up-regulation of CCR7 and CXCR4, but not CD83 and CD86, on mature CB DCs. Cells were pretreated with or without PD98059 for 1 hour. LPS was then added directly into cell culture for 1 day. Cells were harvested and stained with indicated molecules (black histogram) or isotype controls (gray histogram). Expression levels of indicated molecules (percentage of positive cells for CD83, CCR7, and CXCR4, and MFI for CD86) are shown in the upper right corner. (D) Average expression levels plus or minus SEM of 3 to 4 experiments as shown in panel C were presented. (E) PD98059-pretreated mature CB DCs, but not mature AB DCs, functionally migrated at significantly enhanced levels to CCL19 and CXCL12, compared with nontreated cells. Mature CB and AB DCs pretreated with or without inhibitor were assayed side by side in Transwells for chemotaxis toward both CCL19 and CXCL12. The data represent the average level of chemotaxis plus or minus SEM from 3 independent experiments. To minimize sample variation, percentage of migration was calculated relative to percentage of migration of mature AB DCs (defined as 1.0).