Eotaxin-3 is sorted to WPBs and subjected to regulated secretion. (A) HUVECs activated by relevant cytokines and immunostained for IL-8 (clone 4G9) and VWF (top row) or eotaxin-3 (clone 115002) and VWF (bottom row). Corner insets show high magnification of framed areas or negative isotype control (Neg ctr). Original magnification, × 100. (B) Cell-associated levels of IL-8 and eotaxin-3 in IL-1β- or IL-4-activated HUVECs, respectively, were measured by ELISA after incubation without or with PMA (100 ng/mL, 1 hour). 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-4-activated HUVECs incubated without (-) or with (+) PMA (100 ng/mL, 1 hour) before fixation and paired immunostaining for eotaxin-3 (clone 115002) and VWF (original magnification, × 40). The middle panels show high magnification of framed areas in the top panels.