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note
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Each FLAG-CED-4 mutant was cotransfected with AU1-CED-3(C358S) into 293T cells. Cell lysates were immunoprecipitated with antibody to AU1 (anti-AU1) (Babco) and immunoblotted with anti-FLAG. Each CED-4 mutant interacted with CED-3(C358S) as well as wild-type CED-4.
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3543025228
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note
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L, and Apaf-1(1-465) were each fused with a COOH-terminal FLAG epitope tag in pRK5. CED-3(C358S) was also fused to a COOH-terminal AU1 epitope tag in pRK5. Myc - CED-4 and Myc - CED-4 (K165R) were described (3). Myc - Apaf-1(1-465) and Myc - CED-3(C358S) were made in pcDNA3.1(-)/ MycHis (Invitrogen) with a COOH-terminal Myc tag. Authenticity of each construct was confirmed by DNA sequencing.
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3543003591
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note
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6 293T cells [grown in Dulbecco's minimal essential medium supplemented with 10% fetal bovine serum, penicillin-streptomycin (100 U/ml), and glutamine (1 mM)] were plated per 60-mm dish. Twenty-four hours after transfection by the calcium phosphate method, cells were lysed in 300 μl of IP-lysis buffer [50 mM Hepes (pH 7.4), 1% NP-40, 150 mM NaCl, 10% glycerol, 1 mM EDTA, 2 mM dithiothreitol] supplemented with 1 mM phenylmethylsulfonyl fluoride and 1% aprotinin. Extracts (100 μl) were diluted 1:1 in IP-lysis buffer and immunoprecipitated with antibody for 3 hours at 4°C, washed with 600 μl of IP-lysis buffer, and resolved by SDS - polyacrylamide gel electrophoresis (SDS-PAGE).
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3543023396
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note
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We thank F. Chen and H. R. Horvitz for reagents and discussions, P. Svec and A. Darbinian for technical support, and V. M. Dixit, M. Gilman, S. L. Schreiber, and X. Wang for reagents. Supported by the Leukemia Society of America (to X.Y.), the Medical Scientist Training Program (to H.Y.C.), and NIH grant CA51462.
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