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4 fractionation and separation on phosphocellulose, eluting with 200 mM NaCl. These active fractions were pooled and further purified on a Mono-S column (7).
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0023806075
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4. Kinase reactions were terminated by washing the agarose beads twice with 1 ml of TEN [50 mM tris (pH 7.5), 1 mM EDTA, 150 mM NaCI, and 0.5% NP-40] to remove phosphorylated cellular proteins, fractionated on SDS-PAGE, autoradiographed, and stained with Coomassie to ensure that the substrate was not degraded.
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Immunodepletion of GSK-3 activity in 110 μg of whole brain extract was done in 200 μl of TEN, 1 mM DTT, and protease and phosphatase inhibitors (24) with 3 μg of anti-GSK-3a (sheep polyclonal, Upstate Biotechnology), anti-GSK-3β [immunoglobulin G1 (IgG1) monoclonal, Transduction Labs], or both, and 20 μl of protein G-Sepharose at 40°C for 4 hours. The IgGI mouse monoclonal antibody (mAb) M2 (Kodak), sheep polyclonal anti-HIVp17 (NIH), or both were used as control antibodies. The NF-AT kinase assay used 2.5 μl of the supernatant (1.2 μg of protein) (23).
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1842314780
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note
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We thank M. Karin for helpful discussions and sharing unpublished information.
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