Figure 1.

Strategy for searching for substrates for denitrase. To obtain a library of nitrotyrosine-containing proteins, RAW 264.7 cells were treated with peroxynitrite. Sequential extraction according to solubility was performed and each fraction was subjected to 2D membrane assay for denitrase. A pair of samples was applied for 2D electrophoresis and transferred onto poly(vinylidene difluoride) membranes. The membranes were blocked with milk and cut into two pieces of which one was incubated with lysate from LPS-treated RAW 264.7 cells and the other with control solution. The membranes were washed with 1.5 M NaCl, blocked again, and treated with anti-nitrotyrosine Ab. The resulting immunoreactive spots were compared between these membranes, and the spots that showed a decrease of Ab staining intensity after incubation with lysate were assumed to be denitrase substrates and were subjected to peptide sequencing.