Figure 5.

Okadaic acid- and SNP-induced modifications of Ng and activation of CaMKII. Hippocampal slices from WT and KO mice were incubated with 0.5 μM of okadaic acid (A and B) or 0.5 mM SNP (C and D). At timed intervals, samples were taken and tissue extracts used for the measurement of CaMKII activity in the presence or absence of Ca²⁺/CaM. Percent of Ca²⁺/CaM-independent activity was determined. A PO₄-Ng antibody (antibody no. 3615), which is specific for the phosphorylated form, was used for the detection of p-Ng (A Inset) and antibody no. 270 for the detection of total Ng (A Inset) and the reduced (Red) and oxidized (Ox) forms (C Inset). For determination of Ng oxidation (C Inset), slices were homogenized in buffer containing 100 mM iodoacetamide without DTT and electrophoresis carried out under nonreducing condition to preserve the extent of oxidation. Autophosphorylated (p-CaMKII) and total αCaMKII were determined by using commercially available antibodies (B and D). The increase in autophosphorylated CaMKII corresponds well with the increase in the Ca²⁺/CaM-independent activity. The data represent the mean (± SEM) of three separate experiments by using slices from six each of the WT and KO mice.