Impaired Ca²⁺- and cAMP-mediated VWF secretion in EIEE4 BOECs. (A) Healthy control donor and EIEE4 BOEC lysates were separated by SDS-PAGE and were probed with rabbit anti-STXBP1 or mouse anti-actin antibodies followed by incubation with IR dye–labeled donkey anti-rabbit or anti-mouse IgG. (B) Quantification of STXBP1 expression in healthy control donor (black) or EIEE4 BOECs (white) (n = 3). (C) EIEE4 and control donor BOECs were fixed with paraformaldehyde and immunostained for VWF (green) and VE-cadherin (red). Bar represents 10 μm. (D) Endothelial cells were lysed in SF medium containing 1% Triton X-100 and lysates of healthy control donor (black) or EIEE4 BOECs (white) were assayed for VWF content by ELISA. (E) BOECs were incubated for 30 minutes with SF medium (basal), SF medium supplemented with 10 μM forskolin and 100 μM IBMX (FSK), or with 100 μM histamine (HIS). Supernatants were assayed for secreted VWF by ELISA. ***P < .005; n = 3.