Nitration of Tyr-82 is crucial for peroxynitrite- induced internalization. (A) Effect of peroxynitrite (160μM, 1 hour) on the intracellular distribution of a CD40-eGFP hybrid protein expressed by a human endothelial cell line. Column 1, CD40-eGFP–positive cells (green); column 2, cells positive for the plasma membrane marker CD31 (red); and column 3, overlay of columns 1 and 2 with yellow fluorescence indicating localization of CD40 in the plasma membrane. The scale bar represents 10 μm. (B) Mutation of Tyr-82 to alanine abrogates translocation of the hybrid protein. Shown are representative fluorescent cells of at least 3 individual experiments each. The scale bar represents 20 μm (above) and 10 μm (below).