Subcellular localization of Anx A2 and S100A10. (A) HUVECs were incubated in basal medium or forskolin/3-Isobutyl-1-methylxanthine (IBMX)-containing stimulation medium for 20 minutes and subsequently fixed and permeabilized using 0.2% Triton-X100 in 4% paraformaldehyde (PFA)/phosphate-buffered saline. Cells were then stained with anti-AnxA2 (green) and anti-S100A10 (red) antibodies. 4,6 diamidino-2-phenylindole (DAPI) was used to label the nuclei (blue). Bars represent 10 µm. (B) For the quantification of the subcellular localization of AnxA2 and S100A10, signal intensities of both proteins in the cytoplasm and at the plasma membrane of 10 single cells per experiment were analyzed using ImageJ software. Ratios of membrane vs cytosolic intensities were calculated in 3 independent experiments, and the unpaired Student t test was performed to evaluate statistical significance. Bars represent mean ± SEM.