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Volumn 275, Issue 5298, 1997, Pages 394-397

Linkage of g protein-coupled receptors to the MAPK signaling pathway through Pl 3-kinase γ

Author keywords

[No Author keywords available]

Indexed keywords

CARBACHOL; EPIDERMAL GROWTH FACTOR; GUANINE NUCLEOTIDE BINDING PROTEIN; MITOGEN ACTIVATED PROTEIN KINASE; PHOSPHATIDYLINOSITOL 3 KINASE; PROTEIN TYROSINE KINASE; WORTMANNIN;

EID: 0031037296     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.275.5298.394     Document Type: Article
Times cited : (632)

References (29)
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    • note
    • 2, 1 mM dithiotreitol, 2 mM sodium vanadate, 1 mM phenylmethylsulfonylfluoride, aprotinin (20 μg/ml), and leupeptin (20 μg/ml). The lysate was centrifuged at 14,000g for 20 min at 4°C, and proteins were immunoprecipitated and assayed for kinase activity. Equivalent expression of cDNA constructs was verified with the respective antibodies.
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    • note
    • 32P] adenosine triphosphate (ATP) per reaction, 20 μM unlabeled ATP, and myelin basic protein (MBP) (1.5 mg/ml) (Sigma) at 30°C for 30 min. Reactions were terminated by addition of 5X Laemmli buffer. Samples were boiled and proteins were separated by SDS-polyacrylamide gel electrophoresis (PAGE) (12% gel). Phosphorylated MBP was visualized by autoradiography and quantified with either a phosphorimager or a liquid scintillation detector. Parallel samples were immunoprecipitated with antibody to HA and processed for protein immunoblot analysis with a MAPK-specific antiserum.
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    • note
    • Lysates of total cellular protein or anti-HA immunoprecipitates were analyzed by protein immunoblotting after SDS-PAGE with the corresponding rabbit antiserum or mouse mAb. Immunocomplexes were visualized by enhanced chemiluminescence detection (Amersham) with the use of goat antiserum to rabbit or mouse immunoglobulin G coupled to horseradish peroxidase (Cappel, West Chester, PA). Mouse mAbs to the HA epitope 12CA5 were purchased from Babco. Rabbit polyclonal antisera to c-Src, Shc, and Grb2 were purchased from Santa Cruz Laboratories (Santa Cruz, CA) or Upstate Biotechnology (Lake Placid, NY),
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    • note
    • We thank A. Gazit and A. Levitzki for supplying PP1. M.L.I. and R.W. are supported by grants SFB 197 and We 1565/1-2 from the Deutsche Forschungsgemeinschaft.


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