A mammalian telomerase-associated protein

Science

Volumn 275, Issue 5302, 1997, Pages 973-977

  Harrington, Lea ^a   McPhail, Timothy ^b   Mar, Vernon ^b   Zhou, Wen ^b   Oulton, Rena ^a   Bass, Mike B ^b   Arruda, Isabel ^a   Robinson, Murray O ^a  

^a UNIVERSITY OF TORONTO   (Canada)

^b AMGEN INC   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

COMPLEMENTARY DNA; MESSENGER RNA; PROTEIN; PROTEIN ANTIBODY; RNA; TELOMERASE;

AMINO ACID SEQUENCE; ANIMAL CELL; ANIMAL TISSUE; ARTICLE; CONTROLLED STUDY; DNA SEQUENCE; ENZYME ACTIVITY; HUMAN; HUMAN CELL; IMMUNOBLOTTING; IMMUNOPRECIPITATION; MAMMAL; MOLECULAR CLONING; MOUSE; NONHUMAN; NORTHERN BLOTTING; OPEN READING FRAME; PRIORITY JOURNAL; PROTEIN RNA BINDING; SEQUENCE HOMOLOGY; TETRAHYMENA;

AMINO ACID SEQUENCE; ANIMALS; BLOTTING, NORTHERN; CARRIER PROTEINS; CELL LINE; CLONING, MOLECULAR; DNA, COMPLEMENTARY; HUMANS; MICE; MOLECULAR SEQUENCE DATA; PRECIPITIN TESTS; RNA; RNA, MESSENGER; SEQUENCE HOMOLOGY, AMINO ACID; TELOMERASE; TETRAHYMENA; TRANSFECTION; TUMOR CELLS, CULTURED;

ANIMALIA; CILIOPHORA; MAMMALIA; TETRAHYMENA;

EID: 0031036350     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.275.5302.973     Document Type: Article

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50. 5 cells per 100-mm dish in 15 ml of Dulbecco's modified Eagle's medium plus 10% (v/v) fetal calf serum (FCS). For the lipofection, cells were incubated in 6 ml of Optimem I reduced serum medium (Gibco-BRL), 174 μg of Pfx-6 (Invitrogen, San Diego, CA), and 29 μg of full-length, tagged TP1 plasmid DNA (PCR3Myctag3) for 4 hours. The medium was replaced with fresh media containing 10% (v/v) FCS, and the cells were harvested 24 hours later as in (7). Protein lysates were subjected to electrophoresis on 6% (w/v) SDS-polyacrylamide gels and transferred to a nylon membrane. The membrane was incubated with an antibody to Myc (Oncogene Science, San Diego, CA) followed by a horseradish peroxidase-conjugated secondary antibody (Amersham, Arlington Heights, IL) and visualized with the use of an Amersham ECL kit (Amersham). Diethylaminoethyl (DEAE) agarose (Bio-Rad, Hercules, CA) column chromatography was performed as in (21). Approximately 0.5 μg of antibody to human Myc (Oncogene Science) was incubated with 100 to 200 μg of N2A S100 lysate from cells expressing either the Myc-tagged, full-length TP1 (TP1), or with no plasmid (N2A), for 1 hour, followed by incubation with 5 to 10 μl of protein G-Sepharose beads at 4°C for 1 to 3 hours. For the Myc peptide competition, 10 μg of Myc peptide (Oncogene Science) was added before incubation with anti-Myc. The supernatant was removed, and the beads were washed twice with 1 ml of 2,3× hypobuffer (7) containing 10% (v/v) glycerol and 0.1 M NaCI. The lysates (lysate) and immunoprecipitates (IP) were assayed by the telomerase repeat amplification protocol (TRAP) (30). For the anti-peptide immunoprecipitations, sera was adsorbed to 10 μl of protein A-Sepharose beads (Pierce, Rockford, IL), washed with hypobuffer, and incubated with DEAE-purified S100 lysates of NIH 3T3 cells. For peptide competition, 60 μg of each peptide was incubated with the washed protein A beads before the addition of lysate.
51. We thank D. Yeung, L. Antonio, A. Kroll, M. Rihanek, T. Zamborelli A. Hessel, D. Field, J. Hu, and J. Friesen for reagents and technical help; T. Mak, D. Bentley, S. Benchimol, and M. Tyers for helpful discussions and critical reading of the manuscript; and D. Sengupta, M. Wickens, and S. Fields for sharing three-hybrid reagents before publication. The Amgen EST Program is a large interdisciplinary group, who collectively made significant contributions to this work.