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Volumn 275, Issue 5300, 1997, Pages 674-678

NMDA channel regulation by channel-associated protein tyrosine kinase Src

Author keywords

[No Author keywords available]

Indexed keywords

N METHYL DEXTRO ASPARTIC ACID; PROTEIN TYROSINE KINASE;

EID: 0031053363     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.275.5300.674     Document Type: Article
Times cited : (562)

References (37)
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    • Spinal cord membranes were prepared from fetal rats (E17 to E19) with methods adapted from Sheng et al. [M. Sheng, M.-L. Tsaur, Y. N. Jan, L. Y. Jan, Neuron 9, 271 (1992)]. Membrane proteins were solubilized under nondenaturing or denaturing conditions. Nondenaturing conditions: ice-cold 50 mM tris-HCI(pH 7.6) containing 15O mM NaCI, 1% igepal ca630 0.5% sodium deoxycholate, 0.1% SDS, 2 mM EDTA, 1 mM sodium orthovanadate, and the protease inhibitors pepstatin A (20 μg/ml), leupeptin (20 μg/ml), aprotinin (20 μg/ml), and phenylmethylsulfonyl flouride (1 mM). Denaturing conditions: boiling in 1 % SDS followed by fivefold dilution with 50 mM tris-HCI (pH 7.6) containing 190 mM NaCl, 1.25% Triton X-100, 1 mM sodium orthovanadate, 6 mM EDTA, and the protease inhibitors listed above. Solubilized proteins (300 μg) were centrifuged at 14,000g to remove insoluble material and then incubated with anti-NR1 (5 μg) or anti-Src (1:100 dilution) or with control, nonspecific IgG (5 μg) overnight at 4°C. Immune complexes were isolated by the addition of 50 μl of protein G-Sepharose beads followed by incubation for 4 hours at 4°C. Immunoprecipitates were then washed five times with lysis buffer, resuspended in 2 × Laemmli sample buffer, and boiled for 5 min. The samples were subjected to SDS-polyacrylamide gel electrophoresis (PAGE) (10% gel) and transferred to a nitrocellulose membrane. Analysis was performed by repeated stripping of the membrane and successive probing with anti-NR1 (5 μg), anti-Src (1:500 dilution), or anti-Kv3.1 (1:4000 dilution). Sources of antibodies: anti-NR1 (mouse monoclonal anti-NMDAR1): Pharmingen; anti-Src (monoclonal antibody 327): J. Bolen, DNAX, Palo Alto, CA; nonspecific mouse IgG, Calbiochem, San Diego, CA; and anti-Kv3.1 (rabbit antisera): T. Perney, Rutgers University.
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    • note
    • We thank S. Courtneidge, J. Bolen, T. Perney, and R. Wenthold for antibodies; J. F. MacDonald, T. Pawson, and J. C. Roder for critical comments on the manuscript; and Y. De Koninck for software used in analysis. Supported by a grant from the Medical Research Council (MRC) of Canada (M.W.S.) and by the Nicole Fealdman Memorial Fund. M.W.S. is an MRC Scholar, X.-M.Y. is supported by a Fellowship from the Spinal Cord Research Foundation of the Paralyzed Veterans of America, and R.A. is a Fellow of the Rick Hansen Man in Motion Foundation. We thank J. L. Hicks for preparing and maintaining cell cultures and for technical assistance.


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