Munc13-2 localizes to WPB and positively regulates histamine-induced secretion of VWF. (A) HUVEC were fixed and stained for Munc13-2 (yellow) and VWF (magenta) using specific antibodies. Nuclei are shown in cyan. Scale bar, 10 µm. (B) Higher magnification of the boxed region in panel A (merge). Note that almost all VWF-containing WPB are also positive for Munc13-2. As Rab3b and Rab27a are markers for more mature WPB, this could suggest that Munc13-2 might be recruited to WPB in a Rab3b/27a-independent manner. Scale bar, 5 µm. (C-D) Munc13-2 was depleted by transfection with specific siRNAs (siMunc13-2), nontargeting siRNA served as control (siControl). Knockdown efficiency was confirmed by western blot analysis using anti-Munc13-2 antibodies and anti-α-tubulin antibodies as loading control (C). The effect of Munc13-2 depletion on histamine-induced secretion of VWF was measured by an enzyme-linked immunosorbent assay quantifying the amount of VWF released into the cell culture supernatant (D). Note the significant reduction of histamine-evoked VWF secretion after Munc13-2 knockdown. n = 3. Error bars + standard error of the mean. ****P ≤ .0001.