Peroxynitrite and cyclic stretch-mediated membrane depletion and internalization of CD40. (A) After biotinylation of surface proteins for 45 minutes, the cultured endothelial cells were exposed to peroxynitrite (160-200μM) for 1 hour. Thereafter, internalized biotinylated proteins and cells were separated by mild trypsinization and the labeled CD40 collected by immunoprecipitation. Relative amount of label was determined by Western blot analysis with streptavidin-HRP as detection agent. Shown is a representative blot of at least 5 independent experiments. (B) Peroxynitrite treatment (160μM, 1 hour) reduces the abundance of CD40 in the microsomal fraction of cytokine-stimulated (1000 U/mL IFN-γ + 100 U/mL TNF-α, 16 hours) human umbilical vein endothelial cells (HUVEC, left panel) and THP-1 cells (right panel), respectively. Detection was achieved with Abs recognizing the C or N terminus of human CD40 as indicated. L indicates whole-cell lysate; CF, cytosolic fraction; and MF, microsomal fraction. The Western blots are representative of at least 5 individual experiments. A vertical line has been inserted into the left blot to indicate that the whole-cell lysate samples of this experiment were run on a separate gel. Please note that the microsomal fraction naturally contains much less β-actin which nonetheless was rather evenly distributed among these samples. (C) Cyclic stretch (3 or 16 hours, 15% elongation, 0.5 Hz) time-dependently decreases CD40 abundance in the microsomal fraction of the cultured endothelial cells. The exemplary Western blot is representative of 4 independent experiments.