Apoptosis of T cells mediated by Ca2+-induced release of the transcription factor MEF2

Science

Volumn 286, Issue 5440, 1999, Pages 790-793

  Youn, Hong Duk ^a   Sun, Luo ^a   Prywes, Ron ^b   Liu, Jun O ^a  

^a MASSACHUSETTS INSTITUTE OF TECHNOLOGY   (United States)

^b Columbia University ^*  (United States)

Author keywords

[No Author keywords available]

Indexed keywords

CALCINEURIN; CALCIUM; CALMODULIN; STEROID RECEPTOR; T LYMPHOCYTE RECEPTOR; TRANSCRIPTION FACTOR;

APOPTOSIS; ARTICLE; CALCIUM CELL LEVEL; HUMAN; HUMAN CELL; PRIORITY JOURNAL; PROTEIN BINDING; PROTEIN EXPRESSION; SIGNAL TRANSDUCTION; T LYMPHOCYTE; THYMOCYTE;

APOPTOSIS; CALCINEURIN; CALCIUM; CALCIUM SIGNALING; CALMODULIN; CELL LINE; DNA-BINDING PROTEINS; GENE EXPRESSION; GENES, REPORTER; HUMANS; JURKAT CELLS; MYOGENIC REGULATORY FACTORS; PHOSPHOPROTEINS; RECEPTORS, ANTIGEN, T-CELL; RECEPTORS, CYTOPLASMIC AND NUCLEAR; RECEPTORS, STEROID; T-LYMPHOCYTES; TRANSCRIPTION FACTORS; TRANSCRIPTION, GENETIC; TWO-HYBRID SYSTEM TECHNIQUES;

EID: 0033595816     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.286.5440.790     Document Type: Article

References (28)

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22. 2 or 10 mM EGTA. 0.5 μg of GST-Cabin1(2037-2220) was added alone or in combination with 5 μg of CaM to MEF2D-translated products. Reaction mixtures were precipitated using glutathione beads after a 2-hour incubation, separated by 10% SDS-PAGE, and developed by autoradiography.
23. Web figure 2 can be found at www.sciencemag.org/ feature/data/1039959.shl.
24. The pBabeSVCabin1 expression vector was constructed by subcloning the Sal I fragment from pSG-Cabin1, which contains an SV40 promoter and a polyadenylation site flanking the full-length Cabin1 cDNA, into the Sal I site of the pBabe vector [W. S. Pear, G. P. Nolan, M. L. Scott, D. Baltimore, Proc. Natl. Acad. Sci. U.S.A. 90, 8392 (1993)]. DO11.10 cells were transfected with pBabeSVCabin1 and incubated for 24 hours. Transfected cells were treated with puromycin (2 μg/ml) for another 24 hours and dead cells were removed by Ficoll gradient centrifugation. Puromycin-resistant cells were selected by diluting (1:10) transfected cells into puromycin-containing RPMI medium every 3 days until all control DO11.10 cells were dead. The expression of exogenous Cabin1 was confirmed by protein immunoblot using a c-Myc mAb. DNA fragmentation assay was performed as described [K. S. Sellins and J. J. Cohen, J. Immunol. 139, 3199 (1987)].
25. The pBabeSVCabin1 expression vector was constructed by subcloning the Sal I fragment from pSG-Cabin1, which contains an SV40 promoter and a polyadenylation site flanking the full-length Cabin1 cDNA, into the Sal I site of the pBabe vector [W. S. Pear, G. P. Nolan, M. L. Scott, D. Baltimore, Proc. Natl. Acad. Sci. U.S.A. 90, 8392 (1993)]. DO11.10 cells were transfected with pBabeSVCabin1 and incubated for 24 hours. Transfected cells were treated with puromycin (2 μg/ml) for another 24 hours and dead cells were removed by Ficoll gradient centrifugation. Puromycin-resistant cells were selected by diluting (1:10) transfected cells into puromycin-containing RPMI medium every 3 days until all control DO11.10 cells were dead. The expression of exogenous Cabin1 was confirmed by protein immunoblot using a c-Myc mAb. DNA fragmentation assay was performed as described [K. S. Sellins and J. J. Cohen, J. Immunol. 139, 3199 (1987)].
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28. We thank A. Winoto, S. Burakoff, A. Lassar, L. Mathews, E. Seto, and L. Lau for reagents; B. Turk, E. Griffith, A. Mondragon, and other members of the Liu lab for helpful suggestions and critical comments; D. Baltimore and associates for access to a luminometer; and R. Hynes for access to a Gene Pulser. Supported in part by National Institute of General Medical Sciences grant GM55783, the Rita Allen Foundation, and Chiba Prefecture, Japan (J.O.L.) and by a NIH training grant (L.S.).