Fig. 1.
Immunoprecipitation of nitrated proteins from mitochondria. Mitochondrial fractions [total (T), inner membrane (IM), contact sites (CS)] were immunoprecipitated with nitrotyrosine (A and B) or β-subunit antibodies (C and D). Western blots of the immunoprecipitated fractions were then probed for either nitrotyrosine (A and C) or β-subunit (B and D) antibodies. Blots in A and C were blocked with nonfat dry milk (NFDM) with the addition of F(Ab)2, whereas blots in B and D were blocked with NFDM only. This control was done to identify the band appearing at 63 kDa as the heavy chain of immunoglobulins from the immunoprecipitation procedure. E: l-argininne-supplemented rat liver mitochondria isolated from young rats were incubated for 0, 5, 15, 30, and 60 min (●). Samples at each time point were separated by two-dimensional gels, probed for nitrotyrosine, and then stripped and probed for the β-subunit by using Western blots. Control experiments were performed by incubating mitochondria with NG-monomethyl-l-arginine (○), an inhibitor of nitric oxide (NO) synthase. MWM, molecular weight markers.