Time-dependent tyrosine nitration causes CD40 degradation via the proteasome. (A) Colocalization experiment demonstrating the increase in 3-nitrotyrosine content of CD40 protein in endothelial cells treated with MG-132 (10μM, 30-minute preincubation) and exposed to cyclic stretch for 9 hours. Typical Western blot analysis, qualitatively identical results were obtained with at least 4 other batches of cells. (B) Time-dependent changes in the 3-nitrotyrosine content of immunoprecipitated (IP) CD40 protein compared with total CD40 protein (WB) in endothelial cells exposed to cyclic stretch with or without prior MG-132 treatment. Representative Western blot analysis, qualitatively identical results were obtained with at least 2 other batches of cells. (C) Lactacystin (50μM) and epoxomicin (10μM) reverse the stretch-induced decrease (16 hours, 15% elongation, 0.5 Hz) in CD40 protein content (n = 5; *P < .05 vs static control; #P < .05 vs cyclic stretch plus inhibitor). (D) Statistical summary (n = 6-11) of the effect of urate (10μM) on CD40 and VCAM-1 protein abundance in cells subjected to 16 hours of cyclic stretch (*P < .05 vs static control, †P < .05 vs cyclic stretch).