Fig. 5.

LNO₂ covalently modifies PKCζ to induce PKCζ activation. (A) Recombinant PKCζ (50 ng) was used in an in vitro reconstitution activity assay. The recombinant PKCζ was incubated with LA (10 μM) or LNO₂ (10 μM) and the activity of PKC was based on phosphorylation of a specific substrate peptide via transfer of [λ-³²P] ATP by PKC kinase activity. The quantitation of PKCζ activity is shown. Data represent the mean ± SEM (n = 3). *P < 0.01 versus control and LA treatment. (B) Biotinylated LA or LNO₂ at indicated concentrations was incubated with PKCζ in kinase buffer. Labeled proteins were separated by SDS–PAGE and analyzed by Western blot using an anti-biotin primary antibody. A biotin positive band was detected at 66 kDa indicating PKCζ-LNO₂ formation. The data shown are representative Western blot of two separate experiments. (C) These experiments were repeated using non-biotinylated (1 mM) LNO₂ as a competitive reactant to ensure specificity. The data shown are representative of two separate experiments.