AP-1 regulates VWF sorting into WPBs in HUVECs. (A) PCR bands to detect the presence of AP-1 subunits in HUVECs and HT-29 cells. Primers specific for Actin (Act), AP-1μ1A (μ1A), AP-1μ1B (μ1B), and AP-1γ1 (γ1) were used for amplification of cDNA made from extracted RNA from cell lysates. (B) Quantitative reverse-transcription polymerase chain reaction measuring the efficiency of mRNA downregulation of the AP-1μ1A subunit in HUVECs after 2 rounds of nucleofection, with siRNA specifically targeting AP-1μ1A (siAP-1M1), 2 days apart. Mean of 12 experiments with SEM (***P < .0005). (C) Unstimulated VWF secretion on plastic of HUVECs either Mock-nucleofected or nucleofected with siAP-1M1. Cells were treated with either Control (-BFA) or 5 μM of Brefeldin A (+BFA) for 1 hour before and during VWF secretion collection. Results standardized to Mock-BFA samples. Mean of 5 independent experiments with SEM. (***P < .0005). (D) Histamine (100 μM)-stimulated VWF secretion on plastic of HUVECs either Mock-nucleofected or nucleofected with siAP-1M1. Results standardized to Mock samples. Mean of 5 independent experiments with SEM. ***P < .0005.