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Volumn 271, Issue 5247, 1996, Pages 353-356

Rad-dependent response of the chk1-Encoded protein kinase at the DNA damage checkpoint

Author keywords

[No Author keywords available]

Indexed keywords

ATAXIA; ATAXIA TELANGIECTASIA; EUKARYOTA; SCHIZOSACCHAROMYCES POMBE;

EID: 0029664616     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.271.5247.353     Document Type: Article
Times cited : (358)

References (47)
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    • 2] containing protoase inhibitors that is compatible with the activity of lambda protein phosphatase (New England Biolabs) Lysate (25 μg) was incubated with 40 U of phosphatase in a volume of 30 μl for 30 min at 3O°C Control reactions were incubated on ice. Reactions were terminated by addition of 30 μl of 2 × Laemmli sample buffer and heating to 95°C for 5 min.
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    • 3 and 50 mM NaF, and then lysing in the same solution containing protease inhibitors by vortexing with glass beads. After recovery from the beads, the lysate was centrifuged briefly at 5000g to remove unbroken cells For immunoblot analysis, 15 to 25 μg of cell extract was separated on an 8% SDS-polyacrylarnide gel and transferred to nitrocellulose. Blocking of nonspecific sites, antibody incubations, and all washes were performed in PBS containing 1% dried nonfat milk and 0.05% Tween-20. The 12CA5 antibody directed against the HA epitopes was used at a 1:1000 dilution, and the secondary, horseradish peroxidase - coupled goat antibodies to mouse immunoglobulm G (TAGO), at a 1.10,000 dilution. After a final rinse with PBS containing 0 05% Tween-20, immune complexes were detected with the ECL system (Amersham)
    • 3 and 50 mM NaF, and then lysing in the same solution containing protease inhibitors by vortexing with glass beads. After recovery from the beads, the lysate was centrifuged briefly at 5000g to remove unbroken cells For immunoblot analysis, 15 to 25 μg of cell extract was separated on an 8% SDS-polyacrylarnide gel and transferred to nitrocellulose. Blocking of nonspecific sites, antibody incubations, and all washes were performed in PBS containing 1% dried nonfat milk and 0.05% Tween-20. The 12CA5 antibody directed against the HA epitopes was used at a 1:1000 dilution, and the secondary, horseradish peroxidase - coupled goat antibodies to mouse immunoglobulm G (TAGO), at a 1.10,000 dilution. After a final rinse with PBS containing 0 05% Tween-20, immune complexes were detected with the ECL system (Amersham)
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    • We thank members of the Bernards laboratory as well as M Smeets and A. Begg at the Netherlands Cancer Institute for helpful discussions, A. M. Carr for providing strains containing the radΔ alleles, and B Nefsky for critical reading of the manuscript Supported by grants to R B. from the Netherlands Organization for Scientific Research (NWO) and the NIH, and from start-up funds provided to N.W. from UMDNJ-Robert Wood Johnson Medical School
    • We thank members of the Bernards laboratory as well as M Smeets and A. Begg at the Netherlands Cancer Institute for helpful discussions, A. M. Carr for providing strains containing the radΔ alleles, and B Nefsky for critical reading of the manuscript Supported by grants to R B. from the Netherlands Organization for Scientific Research (NWO) and the NIH, and from start-up funds provided to N.W. from UMDNJ-Robert Wood Johnson Medical School


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