H2B exposure on the surface of endothelial cells. Human umbilical vein endothelial cells (HUVECs) were either untreated or treated with annexin V (250 nM) for 48 hr. Cells were surface labeled with biotin, and the biotinylated proteins were isolated using streptavidin-conjugated beads. H2B and p65 (a transcription factor with a cytosolic and nuclear localization) that were bound and eluted from the streptavidin beads were detected by western blotting with a rabbit anti-H2B or rabbit anti-p65. The absence of biotinylated p65 serves as a control for surface labeling of H2B. Band intensities of the western blots were analyzed using Kodak ID 3.6 software, and net intensity (NI) of each band is indicated below each lane. In each set of two lanes, the right-hand lane is in the presence of annexin V and the left-hand lane in its absence. (WCL: whole cell lysates).
