American Society of Hematology

Figure 7

$Figure 7. P-Selectin cell surface delivery and expression levels in CD63-depleted HUVECs. Western blot analysis of whole cell lysates for mock transfected and CD63 depleted cells. Blots were probed with sheep polyclonal anti–human P-selectin and mouse anti–β-tubulin as a loading control. The blot is one representative result from 3 independent experiments. (B) Biotinylation assay to determine surface levels of P-selectin in mock transfected and CD63 depleted cells. IL-4 treated mock-transfected or CD63 siRNA transfected HUVECs were PMA-stimulated for 0-60 minutes followed by incubation with noncell permeable biotin on ice for 30 minutes to label surface proteins. Cells were subsequently lysed and biotinylated proteins pulled down using Neutravidin agarose beads. Protein was eluted by boiling with sample buffer and P-selectin levels analyzed by SDS-PAGE followed by immuno-blotting for P-selectin using sheep polyclonal anti–human P-selectin. Quantification by densitometry analysis (ImageJ) of the immuno-blots are shown. Relative band intensity is shown, normalized to P-selectin surface expression in nonstimulated mock HUVECs. ***P < .001 by 2-way ANOVA. Data are shown as mean ± SD (n = 3-10 for each time point). (C) Sub-cellular fractionation of HUVECs treated with control or CD63-targeted siRNA was performed using a sucrose step-gradient for 2 independent experiments. The graph shows the percentage total VWF across the gradient for both conditions. The major peaks referred to in the text are labeled 1-4. In addition, the Western blot shows the distribution of overexpressed Rab27a-GFP, a marker for WPB. (D) Western blotting of the WPB peak fractions for mock-transfected and CD63 siRNA treated cells was performed to analyze levels of P-selectin. Densitometric analysis of the immuno-blots was carried out to quantify protein expression levels, normalizing to VWF expression. The graph shows the change in P-selectin expression in CD63 depleted cells compared with mock treated, which are set at 1. Two separate biologic repeats were performed for each condition.$

P-Selectin cell surface delivery and expression levels in CD63-depleted HUVECs. Western blot analysis of whole cell lysates for mock transfected and CD63 depleted cells. Blots were probed with sheep polyclonal anti–human P-selectin and mouse anti–β-tubulin as a loading control. The blot is one representative result from 3 independent experiments. (B) Biotinylation assay to determine surface levels of P-selectin in mock transfected and CD63 depleted cells. IL-4 treated mock-transfected or CD63 siRNA transfected HUVECs were PMA-stimulated for 0-60 minutes followed by incubation with noncell permeable biotin on ice for 30 minutes to label surface proteins. Cells were subsequently lysed and biotinylated proteins pulled down using Neutravidin agarose beads. Protein was eluted by boiling with sample buffer and P-selectin levels analyzed by SDS-PAGE followed by immuno-blotting for P-selectin using sheep polyclonal anti–human P-selectin. Quantification by densitometry analysis (ImageJ) of the immuno-blots are shown. Relative band intensity is shown, normalized to P-selectin surface expression in nonstimulated mock HUVECs. ***P < .001 by 2-way ANOVA. Data are shown as mean ± SD (n = 3-10 for each time point). (C) Sub-cellular fractionation of HUVECs treated with control or CD63-targeted siRNA was performed using a sucrose step-gradient for 2 independent experiments. The graph shows the percentage total VWF across the gradient for both conditions. The major peaks referred to in the text are labeled 1-4. In addition, the Western blot shows the distribution of overexpressed Rab27a-GFP, a marker for WPB. (D) Western blotting of the WPB peak fractions for mock-transfected and CD63 siRNA treated cells was performed to analyze levels of P-selectin. Densitometric analysis of the immuno-blots was carried out to quantify protein expression levels, normalizing to VWF expression. The graph shows the change in P-selectin expression in CD63 depleted cells compared with mock treated, which are set at 1. Two separate biologic repeats were performed for each condition.

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