Forskolin-evoked secretion of VWF is inhibited by knockdown of AnxA2 and S100A10. HUVECs were transiently transfected with either nontargeting siRNA duplexes (ct) or duplexes specific for AnxA2 or S100A10. (A-B) Forty-eight hours after transfection, PNSs were prepared and analyzed by immunoblotting for the presence of AnxA2 and S100A10. Probing for α-tubulin and vimentin served as loading controls. Signal intensities were quantified as described in the supplemental Methods, and results from different experiments are summarized in the right panels (n = number of independent experiments). Statistical significance was evaluated by unpaired Student t test. Bars represent mean ± standard error of the mean (SEM). (C-D) Quantification of VWF secretion in response to forskolin from HUVECs depleted of (C) AnxA2 or (D) S100A10. Forty-eight hours following transfection with siRNA duplexes, HUVECs were subjected to agonist treatment of 20 minutes. The amount of VWF released into the cell culture supernatant was then determined as described in Methods. Effects on secretion were quantified in ≥3 independent experiments, and 1-way ANOVA with Bonferroni test was performed to evaluate statistical significance. Bars represent mean ± SEM.