Distribution and release of GROα and MCP-1. (A) IL-1β-activated HUVECs were immunostained for GROα (monoclonal Ab, part 840255 of DuoSet) and VWF (top panels), or for MCP-1 (clone 23007.111) and VWF (bottom panels). Original magnification, × 100. Corner insets show high magnification of framed areas. (B) Cell-associated levels of GROα and MCP-1 in IL-1β-activated HUVECs measured by ELISA after incubation without or with PMA (100 ng/mL, 1 hour). The response of IL-8 toward PMA was examined in parallel wells. Secretory ratios were calculated as explained in the Figure 1 legend, with the dashed line indicating a ratio of 1 and absence of regulated secretion. (C) IL-1β-activated HUVECs incubated without (-) or with (+) PMA (100 ng/mL, 1 hour) before fixation and paired immunostaining as indicated in panel A for GROα (left panels) and VWF (corner insets) or for MCP-1 (right panels) and VWF (not shown). Original magnification, × 40. (D) HUVECs activated by IL-1β were immunostained for GROα (polyclonal, BAF275, or monoclonal Ab, part 840255 of DuoSet) and markers of the endocytic pathway as indicated on the image panels (original magnification, × 100).