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Volumn 276, Issue 5321, 1997, Pages 2042-2045

Regulatory phosphorylation of AMPA-type glutamate receptors by CaM-KII during long-term potentiation

Author keywords

[No Author keywords available]

Indexed keywords

AMPA RECEPTOR; GLUTAMATE RECEPTOR; PROTEIN KINASE;

EID: 0030744875     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.276.5321.2042     Document Type: Article
Times cited : (912)

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    • note
    • P-Thr286, 1 μM microcystine was included in the blotting buffer to inhibit protein phosphatases in the Carnation milk.
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    • note
    • 32P-protein, incubated with GluR1 antibody (13) and protein A-Sepharose at 4°C for 5 hours, and centrifuged. CaM-KII was then immunoprecipitated from the supernatant (20). Immunoprecipitates were run on SDS-polyacrylamide gel electrophoresis (PAGE) and exposed on a Phosphor Screen (Molecular Dynamics) for analyses. The SDS-PAGE of the GluR1 immunoprecipitates were also subjected to protein immunoblot analyses to quantitate the amount of GluR1. Protein content of homogenates was determined by protein assay (Bio-Rad).
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    • note
    • 32P-AMPA-R was cut from the gel, subjected to two digestions with trypsin (16 and 4 hours with 75 μg of trypsin per milliliter), oxidized, and subjected to two-dimensional peptide mapping (cathode on the right) (20).
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    • note
    • 2, and 5 mM Hepes. When added to intracellular solution, activated CaM-KII [truncated at residue 316 and autothiophosphorylated (8)] was at 0.4 μM. Glutamate (10 mM) was delivered to single cells by rapid application (9). Currents were filtered at 2 kHz and digitized at 10 kHz.
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    • 286 antibody
    • 286 antibody.


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