FIGURE 4:

Munc13-4 is required for histamine-induced secretion of VWF. (a) HUVECs were treated with unspecific siRNA as control (siControl) or siRNA targeting Munc13-4 (siMunc13-4), and lysates of the respective cells were subjected to Western blot analysis with anti–Munc13-4 antibodies. Probing with anti–α-tubulin (55 kDa) antibodies served as loading control. Note that endogenous Munc13-4 was successfully depleted. (b) ELISA-based VWF secretion assay. HUVECs were transfected with siControl or Munc13-4 targeting siRNA (siMunc13-4) plus expression vectors encoding YFP (control), YFP-Munc13-4, or GFP-Munc13-4(Δ280-285), which were both rendered insensitive to the specific Munc13-4 siRNA (see Materials and Methods). In each case, the amount of VWF secreted into the cell culture supernatant was measured by ELISA. Means of at least five independent experiments that were tested for statistical significance by one-way ANOVA with Tukey’s test (ns, not statistically significant, *p ≤ 0.05, **p ≤ 0.01, ****p ≤ 0.0001). Bars represent mean ± SEM. Numbers of independent experiments: siControl plus YFP or Munc13-4, eight; siControl plus Δ280-285, seven; siMunc13-4 plus YFP or Munc13-4, six; siMunc13-4 plus Δ280-285, five.