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. 2008 Oct 14;6(10):e252. doi: 10.1371/journal.pbio.0060252

Figure 6. PARylation of ISWI Inhibits Nucleosome Binding In Vitro.

Figure 6

Gel retardation assays of (A) mixed-length poly-nucleosomes purfied from chicken erythrocyte or in vitro assembled monoucleosomes (B) after incubation with increasing amounts of ISWI, in the presence or absence of PARP and in the presence or absence of PARP plus its competitive inhibitor 3-AB. For purified nucleosomes, 125 ng of a mixed length poly-nucleosome fraction were incubated with 2, 4, 8, and 16 nmol of ISWI, 0.4 nmol of PARP and 5 mM 3-AB. For reconstituted recombinant nucleosomes, 0.25 nmol of mononucleosomes were incubated with 0.5, 1, 2, and 4 nmol of ISWI, 0.2 nmol of PARP, and 2.5 mM 3-AB.

Activating DNA together with increasing amounts of ISWI were tested for the ability to shift (C) purified polynucleosomes and (D) recombinant mononucleosomes before and after incubation with PARP or PARP and 3-AB. Sixteen, and 32 nmol of ISWI, 0.4 nmol of PARP, and 40 mM 3-AB were used for purified nucleosomes, while 4 and 8 nmol of ISWI, 0.2 nmol of PARP, and 20 mM 3-AB were used for reconstituted recombinant nucleosomes. Numbers indicate mass ratio of ISWI over purified polynucleosomes (ISWI/Poly) or over in vitro assembled mononucleosomes (ISWI/Mono).