Figure 2.

p55 is a subunit of NURF. (A) p55 copurifies with active NURF fractions. Western blot of chromatographic fractions containing equivalent NURF activity. NE, nuclear extract; BR, DE52/BioRex; Q, Q Sepharose; HAP, hydroxylapatite; SS, single-stranded DNA cellulose; P11, phosphocellulose P-11; Gly, glycerol gradient. The blot was probed with antibodies to p55. Antibody specificity was demonstrated by the inhibition of immunoreactivity by the inclusion of purified recombinant p55 protein with anti-p55. (B) p55 coimmunoprecipitates with ISWI. Western blot of proteins coimmunoprecipitating with p55 and ISWI. Nuclear extracts were incubated with affinity-purified anti-ISWI (α-ISWI) (Left) or anti-p55 serum (α-p55) (Right), preimmune sera, or protein G beads alone. Immunoprecipitates were analyzed by SDS-PAGE and Western blotting using α-p55 and α-ISWI, respectively. (C) Quantitative immunodepletion of ISWI and p55 from a NURF fraction (P-11 equivalent; ref. 13) by affinity-purified anti-p55 and control anti-chicken IgG. Western blot analysis of the supernatants (Sup) and pellet (Beads) showing ISWI and p55. The SDS-PAGE mobility of p55 immunoprecipitated by α-p55 beads is slightly increased by the presence of comigrating IgG. (D). GAGA transcription factor-mediated nucleosome disruption on reconstituted hsp70 plasmid chromatin, using NURF (P-11 fraction) and the same fraction immunodepleted with α-p55 and control anti-chicken IgG (α-IgG). Southern blot showing nucleosome disruption as assayed by MNase digestion (3- and 15-min digestion points for each chromatin sample); the blot was hybridized with radiolabeled oligonucleotide corresponding to the hsp70 promoter (−113 to −142) or downstream sequences (+1803 to +1832) as described (13). The position of mononucleosomal DNA is indicated by the dot. NURF activity is gauged by the relative loss of the mononucleosomal DNA fragment and concomitant appearance of subnucleosomal DNA fragments.