Figure 1.

Detection of phosphorylation of nNOS at S1412 with a phosphopeptide antiserum specific for S1412. A, Schematic diagram of nNOS showing the NOSox and NOSred modules, the CaM-binding linker, and the location of the phosphorylation sites at S1412 and S847. The AI insert, the C-terminal tail (CT), and the FMN domain of NOS are also illustrated. B, Autoradiogram of SDS-PAGE of ³²P-nNOS labeled in the presence of γ³²P ATP and Akt. C, Western blots showing the specificity of the antibody against S1412-PO₄ for nNOS phosphorylated by Akt. NOS was incubated with Akt plus nonradioactive ATP, or without any enzyme as a control. Lane 1, No enzyme, control; lane 2, Akt. A Western blot for total nNOS showing equivalent levels of nNOS protein loading. The specificity of the antibody to S1412-PO₄ for phosphorylated nNOS was demonstrated further using extracts from an adult rat brain. D, Western blots of a rat whole-brain lysate, fractionated by 10% SDS-PAGE, with antibody to S1412-PO₄ and polyclonal nNOS antibody (Transduction Laboratories, Lexington, KY).