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cells were trypsinized, counted and washed once in PBS. They were resuspended in buffer1 (0.32 M sucrose, 10 mM Tris, pH8.0, 3 mM CaCl2, 2 mM Mg-acetate, 0.1 mM EDTA, 0.5% NP40, 1 mM DTT, 0.5 mM PMSF; 1 × 107cells/400μl buffer) and sonified on ice (sonifier UP200S, dr.hielscher, 40 s, amplitude 50%, cycle 0.5). This step was repeated until no more intact cells were observed by light microscopy. The resulting homogenate was centrifuged at 500 g for 5 minutes, washed twice in 1 ml buffer 1 without NP40 and centrifuged again. The resulting pellet contains purified nuclei. For partial extraction of the nucleoplasm according to [37], nuclei were resuspended in buffer 2 (20 mM Hepes, pH 7.9, 1.5 mM MgCl2, 20 mM KCl, 0.2 mM EDTA, 25%v/v glycerine, 0.5 mM DTT, 0.5 mM PMSF; nuclei of 107cells/30 μl buffer) and incubated on ice for 30 minutes. An equal volume of buffer 3 was added in aliquots (20 mM Hepes, pH 7.9, 1.5 mM MgCl2, 800 mM KCl, 0.2 mM EDTA, 25%v/v glycerine, 0.5 mM DTT, 0.5 mM PMSF, 1% NP40, protease inhibitor cocktail (Sigma)) and the solution was incubated while shaking for further 30 minutes on ice. Then the solution was centrifuged for 15 minutes at 14.000 g, the resulting supernatant was used as nucleoplasm, and the pellet was washed once and adjusted to an equal volume with PBS. Nucleoplasm and nuclear pellet were subjected to SDS-PAGE and Western blotting."}

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They were resuspended in buffer1 (0.32 M sucrose, 10 mM Tris, pH8.0, 3 mM CaCl2, 2 mM Mg-acetate, 0.1 mM EDTA, 0.5% NP40, 1 mM DTT, 0.5 mM PMSF; 1 × 107cells/400μl buffer) and sonified on ice (sonifier UP200S, dr.hielscher, 40 s, amplitude 50%, cycle 0.5). This step was repeated until no more intact cells were observed by light microscopy. The resulting homogenate was centrifuged at 500 g for 5 minutes, washed twice in 1 ml buffer 1 without NP40 and centrifuged again. The resulting pellet contains purified nuclei. For partial extraction of the nucleoplasm according to [37], nuclei were resuspended in buffer 2 (20 mM Hepes, pH 7.9, 1.5 mM MgCl2, 20 mM KCl, 0.2 mM EDTA, 25%v/v glycerine, 0.5 mM DTT, 0.5 mM PMSF; nuclei of 107cells/30 μl buffer) and incubated on ice for 30 minutes. An equal volume of buffer 3 was added in aliquots (20 mM Hepes, pH 7.9, 1.5 mM MgCl2, 800 mM KCl, 0.2 mM EDTA, 25%v/v glycerine, 0.5 mM DTT, 0.5 mM PMSF, 1% NP40, protease inhibitor cocktail (Sigma)) and the solution was incubated while shaking for further 30 minutes on ice. Then the solution was centrifuged for 15 minutes at 14.000 g, the resulting supernatant was used as nucleoplasm, and the pellet was washed once and adjusted to an equal volume with PBS. Nucleoplasm and nuclear pellet were subjected to SDS-PAGE and Western blotting."}

{"project":"craft-ca-core-dev","denotations":[{"id":"T10459","span":{"begin":0,"end":14},"obj":"CL:0000010"},{"id":"T10460","span":{"begin":106,"end":113},"obj":"CHEBI:17992"},{"id":"T10461","span":{"begin":121,"end":125},"obj":"CHEBI:9754"},{"id":"T10462","span":{"begin":139,"end":144},"obj":"CHEBI:3312"},{"id":"T10463","span":{"begin":151,"end":161},"obj":"CHEBI:62964"},{"id":"T10464","span":{"begin":181,"end":185},"obj":"CHEBI:63016"},{"id":"T10465","span":{"begin":192,"end":195},"obj":"CHEBI:18320"},{"id":"T10466","span":{"begin":204,"end":208},"obj":"CHEBI:8102"},{"id":"T10467","span":{"begin":510,"end":514},"obj":"CHEBI:63016"},{"id":"T10468","span":{"begin":577,"end":583},"obj":"GO:0005634"},{"id":"T10469","span":{"begin":615,"end":626},"obj":"GO:0005654"},{"id":"T10470","span":{"begin":646,"end":652},"obj":"GO:0005634"},{"id":"T10471","span":{"begin":689,"end":694},"obj":"CHEBI:46756"},{"id":"T10472","span":{"begin":711,"end":716},"obj":"CHEBI:6636"},{"id":"T10473","span":{"begin":724,"end":727},"obj":"CHEBI:32588"},{"id":"T10474","span":{"begin":749,"end":758},"obj":"CHEBI:17754"},{"id":"T10475","span":{"begin":767,"end":770},"obj":"CHEBI:18320"},{"id":"T10476","span":{"begin":779,"end":783},"obj":"CHEBI:8102"},{"id":"T10477","span":{"begin":785,"end":791},"obj":"GO:0005634"},{"id":"T10478","span":{"begin":912,"end":917},"obj":"CHEBI:46756"},{"id":"T10479","span":{"begin":934,"end":939},"obj":"CHEBI:6636"},{"id":"T10480","span":{"begin":948,"end":951},"obj":"CHEBI:32588"},{"id":"T10481","span":{"begin":973,"end":982},"obj":"CHEBI:17754"},{"id":"T10482","span":{"begin":991,"end":994},"obj":"CHEBI:18320"},{"id":"T10483","span":{"begin":1003,"end":1007},"obj":"CHEBI:8102"},{"id":"T10484","span":{"begin":1012,"end":1016},"obj":"CHEBI:63016"},{"id":"T10485","span":{"begin":1037,"end":1045},"obj":"CHEBI:60004"},{"id":"T10486","span":{"begin":1063,"end":1071},"obj":"CHEBI:75958"},{"id":"T10487","span":{"begin":1140,"end":1148},"obj":"CHEBI:75958"},{"id":"T10488","span":{"begin":1231,"end":1242},"obj":"GO:0005654"},{"id":"T10489","span":{"begin":1317,"end":1328},"obj":"GO:0005654"},{"id":"T10490","span":{"begin":1333,"end":1340},"obj":"GO:0005634"},{"id":"T10491","span":{"begin":1366,"end":1369},"obj":"CHEBI:8984"}],"text":"Cultured cells were trypsinized, counted and washed once in PBS. They were resuspended in buffer1 (0.32 M sucrose, 10 mM Tris, pH8.0, 3 mM CaCl2, 2 mM Mg-acetate, 0.1 mM EDTA, 0.5% NP40, 1 mM DTT, 0.5 mM PMSF; 1 × 107cells/400μl buffer) and sonified on ice (sonifier UP200S, dr.hielscher, 40 s, amplitude 50%, cycle 0.5). This step was repeated until no more intact cells were observed by light microscopy. The resulting homogenate was centrifuged at 500 g for 5 minutes, washed twice in 1 ml buffer 1 without NP40 and centrifuged again. The resulting pellet contains purified nuclei. For partial extraction of the nucleoplasm according to [37], nuclei were resuspended in buffer 2 (20 mM Hepes, pH 7.9, 1.5 mM MgCl2, 20 mM KCl, 0.2 mM EDTA, 25%v/v glycerine, 0.5 mM DTT, 0.5 mM PMSF; nuclei of 107cells/30 μl buffer) and incubated on ice for 30 minutes. An equal volume of buffer 3 was added in aliquots (20 mM Hepes, pH 7.9, 1.5 mM MgCl2, 800 mM KCl, 0.2 mM EDTA, 25%v/v glycerine, 0.5 mM DTT, 0.5 mM PMSF, 1% NP40, protease inhibitor cocktail (Sigma)) and the solution was incubated while shaking for further 30 minutes on ice. Then the solution was centrifuged for 15 minutes at 14.000 g, the resulting supernatant was used as nucleoplasm, and the pellet was washed once and adjusted to an equal volume with PBS. Nucleoplasm and nuclear pellet were subjected to SDS-PAGE and Western blotting."}