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. 2002 Aug;22(15):5626–5637. doi: 10.1128/MCB.22.15.5626-5637.2002

FIG. 2.

FIG. 2.

Reconstitution of chromatin on templates containing HNF-4 cognate sites. (A) Purified preparations of the indicated proteins used for reconstituting chromatin on plasmid pA4xMLΔ53 were analyzed by SDS-PAGE and stained with Coomassie brilliant blue (lanes 1 and 5) or silver (lanes 2 to 4). Lane 1, HeLa cell histones, 1.5 μg; lane 2, baculovirus-expressed Acf-1, 200 ng; lane 3, baculovirus-expressed ISWI, 200 ng; lane 4, bacterially expressed NAP-1, 200 ng; lane 5, baculovirus-expressed p300, 1 μg. (B) DNA supercoiling assay for chromatin assembly on plasmid pA4xMLΔ53. Plasmid DNA, treated as described, was electrophoresed on agarose gels and stained with ethidium bromide. Lane 1, purified plasmid prior to assembly; lane 2, topoisomerase I-relaxed plasmid; lane 3, plasmid after chromatin assembly (and deproteinization). Supercoiled (sc) and relaxed (rel) DNAs are marked. (C) MNase digestion assay for chromatin assembly. After assembly into chromatin, plasmid pA4xMLΔ53 was digested with MNase, deproteinized, and analyzed by agarose gel electrophoresis. A 123-bp ladder (M) was used as a size marker.