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14
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2642620264
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note
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2 for 1 hour at 4°C. Bound proteins were eluted with 15 mM EGTA, fractionated on a Mono Q column (linear gradient of 0 to 500 mM NaCl), and subjected to immunoblot analysis with antibodies to PP2A subunits and CaMKIV (20). Proteins in the fractions containing the peak of immunoreactivity for CaMKIV and PP2A were precipitated with 50% ammonium sulfate, resuspended, and fractionated by Superdex-200 gel filtration.
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15
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2642651941
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note
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Samples were separated on SDS-polyacrylamide gels (10%) and transferred to a nitrocellulose membrane in 10 mM CAPS (3-cyclohexylamino-1-propane sulfonic acid) containing 10% methanol for 1 hour at 1 A. Proteins on the membrane were visualized with Ponceau S followed by washing in TTBS [25 mM tris-HCl (pH 7.4), 137 mM NaCl, 3 mM KCl, and 0.2% Tween-20]. After incubation with 2% nonfat milk in TTBS for 1 hour, membranes were incubated with affinity-purified polyclonal antibodies to the A and Bα subunits of PP2A (1:500) or with monoclonal antibodies to the C subunit of PP2A (1:5000) or to CaMKIV (1:2000 for 1 hour). Membranes were then incubated with alkaline phosphatase-or horseradish peroxidase-conjugated secondary antibodies for 1 hour, and bound antibodies were visualized by colorimetric detection or chemiluminescence. Calmodulin overlays were done as described for the immunoblot analysis, except that biotinylated calmodulin (1:1500) was substituted for the primary antibody and alkaline phosphatase-conjugated streptavidin (1:10,000) was substituted for the secondary antibody. Assessment of CaMKIV and PP2A molar stoichiometry was performed by preparing a standard curve of purified CaMKIV and PP2A C subunit and comparing the immunoblot intensities with partially purified CaMKIV-PP2A samples.
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17
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2642646284
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note
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Soluble extract was prepared from one rat brain by homogenization in phosphate-buffered saline (PBS) containing 1 mM PMSF, leupeptin (10 μg/ml), soybean trypsin inhibitor (20 μg/ml), pepstatin (2 μg/ml), and 2 mM benzamidine, and centrifugation at 30,000g for 45 min. Approximately 1 mg of extract was incubated with purified GSTfusion protein (10 μg), or buffer, for 3 hours at 4°C. Glutathione-Sepharose (40 μl of a 1:1 slurry) was added and the incubation was continued 1 hour. The beads were collected and washed six times (5 min per wash) with PBS. Bound proteins were eluted with 20 mM glutathione in PBS at room temperature. Eluates were subjected to immunoblotting.
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19
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0028089860
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C. MacKintosh and R. W. MacKintosh, Trends Biochem. Sci. 19, 444 (1994). Rat brain soluble extracts were prepared as described (14). Jurkat T cell extracts were prepared by homogenization in buffer A containing 0.5% Triton X-100 and centrifugation for 15 min at 13,000g. Microcystin-Sepharose (40 μl of a 1:1 slurry) in the absence or presence of 1 μM microcystin was added to the supernatant and the samples incubated overnight at 4°C. The beads were washed six times (5 min per wash) with buffer A, and bound proteins eluted with Laemmli sample buffer were subjected to immunoblot analysis.
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R. P. S. Kwok et al., ibid. 370, 223 (1994).
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2642678502
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note
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2. The calmodulin-Sepharose was extensively washed, and bound proteins eluted in homogenization buffer containing 15 mM EGTA were subjected to immunoblot analysis.
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37
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2642611152
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6 cells per 0.2 ml of RPMI medium with fetal calf serum (10%), mixed with the indicated plasmids, and transfected by electroporation with a Bio-Rad Gene Pulser (250 V, 960 μF).
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38
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2642701765
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Supported by NIH grants GM51366 (B.E.W.), GM33976 and HD07503 (A.R.M.), and NRSA F32 AI09258 (K.A.A.); by grants from the Vanderbilt Diabetes Research and Training Center (DK20593), Cancer Center (CA68485), and Center for Molecular Neuroscience (MH19732); and by the Keck Foundation (Duke University). B.E.W. is the recipient of a Faculty Development Award from the Pharmaceutical Research and Manufacturers of America Foundation. We thank R. Colbran, J. Scott, L. Limbird, D. Lovinger, L. Kerr, and S. Shenolikar for critical discussions of the data and manuscript.
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