AnxA2 containing a phosphomimicking mutation at Ser-11 does not promote forskolin-induced VWF secretion. Forty-eight hours after transfection with AnxA2 siRNA or nontargeting siRNA (ct RNAi), HUVECs were transfected a second time either with siRNA specific for AnxA2, control siRNA, or with AnxA2 siRNA together with siRNA-insensitive AnxA2 mutant constructs (S11A or S11D). Rescue of AnxA2 protein levels following expression of the (A) AII-Rr-S11A or (B) AII-Rr-S11D constructs was verified by immunoblotting using polyclonal rabbit anti-AnxA2 antibodies for the detection of endogenous and exogenous AnxA2 (total) or monoclonal mouse anti-AnxA2 antibodies (clone H28) for the detection of only ectopically expressed S11A- and S11D-AnxA2 (ect.). Probing with anti–α-tubulin antibodies was used as internal loading control. (C-D) HUVECs transiently transfected with AnxA2 siRNA plus the respective rescue plasmids ([C] AII-Rr-S11A and [D] AII-Rr-S11D) were stained with (left) anti-AnxA2 antibodies (clone H28 to label ectopically expressed AnxA2) and (right) anti-S100A10 antibodies directly coupled to Alexa Fluor 594. Nuclei were labeled with DAPI (blue). Note the restoration of endogenous S100A10 levels in cells ectopically expressing AII-Rr-S11A but not in cells expressing AII-Rr-S11D. Scale bars represent 10 µm. (E-F) HUVECs transfected with siRNA and the respective rescue constructs were subjected to forskolin/IBMX treatment, and VWF secretion was analyzed by ELISA. The effect on secretion was quantified in (C) 9 and (D) 11 independent experiments. One-way ANOVA with Bonferroni test was used for the quantification of statistical significance. Bars represent mean ± SEM.