The phosphomimicking S11D mutation in AnxA2 attenuates the interaction between AnxA2 and S100A10 in HUVECs. HUVECs were transiently transfected with WT-, S11A-, or S11D-AnxA2, respectively. Forty-eight hours after transfection, PNSs were prepared and subjected to immunoprecipitation using mouse monoclonal antibodies only recognizing the ectopically expressed AnxA2 derivatives (clone H28; supplemental Methods). The amount of AnxA2 derivative (ect. AnxA2) and S100A10 present in the PNSs and the immunoprecipitates was quantified as described in supplemental Methods. For quantification, AnxA2 levels in IP fractions were normalized to the respective level in the PNS fraction (input), and the amount of coprecipitated S100A10 was calculated as percentage of precipitated AnxA2 (bar diagram). Statistical significance of the results obtained from 3 independent experiments was evaluated by 1-way ANOVA with the unpaired Student t test. Bars represent mean ± SEM.