Impaired Ca2+- and cAMP-mediated VWF secretion after STXBP1 depletion. (A) HUVECs were nucleofected with siRNA oligos directed against STXBP1 (siSTXBP1) or with nonhybridizing control oligos (siCTRL) and were assayed 48 hours after nucleofection. 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 control cells (black) or after STXBP1 depletion (white) (n = 3). (C) Endothelial cells were lysed in SF medium containing 1% Triton X-100 and were assayed for VWF content by ELISA (n = 3). (D) Endothelial cells 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).
