CD40 nitration enhances susceptibility to degradation by the 20S proteasome. (A) Effect of authentic peroxynitrite (160μM, 1 hour) and the proteasomal inhibitor lactacystin on the CD40 protein content of endothelial cells cultured under static conditions (n = 5; *P < .05 vs medium control), and representative Western blot analysis. Detection of CD40 tyrosine nitration (N-Tyr) with (B) immunoprecipitated native CD40 protein and (C) a recombinant CD40/Fc chimeric protein consisting of the extracellular domain of human CD40 and the Fc region of human IgG1. Nitration was achieved with the indicated concentrations of SIN-1 (peroxynitrite donor) and authentic peroxynitrite (1-hour exposure at 37°C). Representative Western blot analyses of at least 6 independent experiments. (D) Proteasome-dependent degradation of nitrated CD40 (N-Tyr) in a cell-free system. Native immunoprecipitated CD40 protein was incubated for 2 hours with 0.1 mg/mL recombinant 20S or 26S proteasome in the absence or presence of lactacycstin (50μM). Exemplary Western blot analysis, 2 repeat experiments produced the same result. (E) Time course of the degradation of nitrated CD40 protein (N-Tyr) by the 20S proteasome. Recombinant CD40/Fc chimeric protein was incubated with the recombinant 20S proteasome (0.1 mg/mL) for the indicated periods (n = 3; *P < .05 vs unmodified CD40).