Figure 4

In vitro phosphorylation of CaMKK substrates in rat brain extract with Mg-GTP. A. Rat brain extract (5.6 mg/ml) was incubated without (-) or with recombinant CaMKKα (α) or β (β) isoform (2 μg) in the absence (-) or presence (+) of 0.1 mM GTP at 30°C for 1 min in a solution containing 50 mM HEPES (pH 7.5), 10 mM Mg(CH₃COO)₂, 2 mM DTT, 2 mM CaCl₂ and 4.5 μM CaM and indicated concentrations (0, 1, and 10 μg/ml) of STO-609, followed by western blot analysis with anti-phospho-CaMKI antibody. B. Rat brain extract (6.6 mg/ml) was incubated without (-) or with 1 mM GTP (+) at 30°C for 30 min in the presence of either 2 mM EGTA (-) or 2 mM CaCl₂/11 μM CaM (+) in a solution containing 50 mM HEPES (pH 7.5), 10 mM Mg(CH₃COO)₂, 2 mM DTT and indicated concentrations (0, 1, or 10 μg/ml) of STO-609, followed by western blot analysis with anti-phospho-threonine antibody. The asterisk and arrowheads indicate rat brain proteins whose phosphorylation was induced by Ca²⁺/CaM. Results are representative of at least three independent experiments.