Figure 3: AnxA8 depletion leads to increased internalization of P-selectin from the cell surface.

(a) HUVEC transfected with control (upper panel) or anxA8-specific siRNA (lower panel) were co-stained with antibodies recognizing P-selectin (red) and VWF (green). Draq5 was used to label the nuclei. Colocalization coefficients (of VWF with P-selectin) from at least 23 images from five independent experiments were determined. Data are means±s.e.m. Scale bars, 10 μm. *P<0.05; unpaired Student’s t-test. (b) Non-transfected HUVEC (NT) and HUVEC transfected with either non-targeting (siCt) or anxA8-specific siRNA (siA8) were cultivated for 48 h and then incubated in media containing antibodies against P-selectin for 24 h, fixed, permeabilized, incubated with the respective secondary antibodies and co-stained with antibodies against VWF. Scale bars, 10 μm. Colocalization coefficients of VWF with P-selectin antibodies from at least 13 individual cells from three independent experiments were determined as described in Methods. Statistical analysis was performed by one-way ANOVA followed by Tukey’s multiple comparison test. P>0.05=NS. (c) HUVEC transiently transfected with either non-targeting siRNA or anxA8-specific siRNA were allowed to internalize FITC-coupled anti-P-selectin antibodies at 37 °C for 30 min and subsequently analysed by flow cytometry. Relative fluorescence of internalized P-selectin was quantified and statistically analysed by unpaired Student’s t-test. *P<0.05; data are means±s.e.m. from four independent experiments (10,000 cells per experiment).