Figure 5
Figure 5. AP-1 regulates basolateral sorting of VWF constitutive carriers. (A) Unstimulated VWF secretion from HUVECs grown on Transwell membranes either Mock-nucleofected or nucleofected with siRNA specifically targeting AP-1μ1A (siAP-1M1). Results standardized to Mock samples. Mean of 5 independent experiments with SEM. *P < .05, **P < .005. (B) Unstimulated VWF secretion from HUVECs grown on Transwell membranes 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 samples. Mean of 3 independent experiments with SEM. **P < .005. (C) lumGFP secretion on Transwell membranes. HUVECs were either Mock-nucleofected or nucleofected with siRNA specifically targeting AP-1μ1A (siAP-1M1), and lumGFP construct was added during the second round of nucleofection and seeded on Transwell membranes for 30 hours before secretion collection. Cells were treated with either Control (-BFA) or 5 μM of Brefeldin A (+BFA) for 1 h before and during secretion collection in both chambers. Amount of lumGFP secreted expressed as a percent of total GFP in each sample (secreted plus lysate). Mean of 3 replicates with SD. ***P < .0005. (D) Histamine (100 μM) stimulated VWF secretion from HUVECs grown on Transwell membranes either Mock-nucleofected or nucleofected with siRNA, specifically targeting AP-1μ1A (siAP-1M1). Results standardized to Mock samples. Mean of 7 independent experiments with SEM. (E) Representative multimer pattern of secreted VWF collected from unstimulated or 100 μM Histamine–stimulated HUVECs either Mock-nucleofected or nucleofected with siAP-1M1. Unstimulated secretion was collected for 1 hour and histamine-stimulated secretion was collected for 30 minutes. The same units of VWF (as measured by ELISA) were loaded for all samples. VWF ELISA quantification corresponding to these samples are shown in (A) and (D). A, apical chamber; B, basolateral chamber. Lanes 1-4, unstimulated releasate; lanes 5-8, histamine-stimulated releasate.

AP-1 regulates basolateral sorting of VWF constitutive carriers. (A) Unstimulated VWF secretion from HUVECs grown on Transwell membranes either Mock-nucleofected or nucleofected with siRNA specifically targeting AP-1μ1A (siAP-1M1). Results standardized to Mock samples. Mean of 5 independent experiments with SEM. *P < .05, **P < .005. (B) Unstimulated VWF secretion from HUVECs grown on Transwell membranes 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 samples. Mean of 3 independent experiments with SEM. **P < .005. (C) lumGFP secretion on Transwell membranes. HUVECs were either Mock-nucleofected or nucleofected with siRNA specifically targeting AP-1μ1A (siAP-1M1), and lumGFP construct was added during the second round of nucleofection and seeded on Transwell membranes for 30 hours before secretion collection. Cells were treated with either Control (-BFA) or 5 μM of Brefeldin A (+BFA) for 1 h before and during secretion collection in both chambers. Amount of lumGFP secreted expressed as a percent of total GFP in each sample (secreted plus lysate). Mean of 3 replicates with SD. ***P < .0005. (D) Histamine (100 μM) stimulated VWF secretion from HUVECs grown on Transwell membranes either Mock-nucleofected or nucleofected with siRNA, specifically targeting AP-1μ1A (siAP-1M1). Results standardized to Mock samples. Mean of 7 independent experiments with SEM. (E) Representative multimer pattern of secreted VWF collected from unstimulated or 100 μM Histamine–stimulated HUVECs either Mock-nucleofected or nucleofected with siAP-1M1. Unstimulated secretion was collected for 1 hour and histamine-stimulated secretion was collected for 30 minutes. The same units of VWF (as measured by ELISA) were loaded for all samples. VWF ELISA quantification corresponding to these samples are shown in (A) and (D). A, apical chamber; B, basolateral chamber. Lanes 1-4, unstimulated releasate; lanes 5-8, histamine-stimulated releasate.

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