1. Volume 8, Issue 3, Pages 531-543 (September 2001)
Dual Functions of Largest NURF Subunit NURF301 in Nucleosome Sliding and Transcription Factor Interactions 
Hua Xiao, Raphael Sandaltzopoulos, Hih-Min Wang, Ali Hamiche, Ryan Ranallo, Kyu-Min Lee, Dragony Fu, Carl Wu 
Molecular Cell 
Volume 8, Issue 3, Pages (September 2001)
DOI: /S (01)

2. Figure 1 cDNA Cloning of Drosophila NURF301
(A) Schematic representation of NURF301 and ACF1 (Ito et al., 1999). Shown above are sequences matching NURF301 tryptic peptides; arrows indicate peptide positions.
(B) Sequence alignment of human HMGI (Friedmann et al., 1993) with Chironomus HMGI/Y (Claus et al., 1994) and NURF301. Asterisks indicate sites of serine/threonine phosphorylation and lysine acetylation (Reeves and Beckerbauer, 2001).
(C) Northern blot analysis of NURF301 mRNA. 1 μg of Drosophila embryonic poly(A) RNA was resolved on a 0.8% agarose, formaldehyde-TBE gel, blotted, and probed with a 32P-labeled NURF301 cDNA clone.
(D) Antibody specificity and co-IP of NURF301 with ISWI. Western blotting of E. coli extracts containing GST-NURF301 fusion proteins (∼2 ng). The protein blot was probed with a polyclonal rabbit antibody against a peptide from amino acids 1141–1161 of NURF301 (lanes 1 and 2). Anti-301 peptide antibody reacts specifically with the largest subunit of NURF purified from Drosophila embryo extracts (glycerol gradient step) (Sandaltzopoulos et al., 1999) (lane 3). Co-IP of NURF301 and ISWI using preimmune and anti-ISWI antibodies, respectively (lanes 4 and 5)
Molecular Cell 2001 8, DOI: ( /S (01) )

3. Figure 2 Reconstitution of NURF
(A) SDS-PAGE of purified recombinant NURF subunits. SYPRO orange staining of FLAG-NURF301, FLAG–ISWI, c-myc-NURF55, and AU1-NURF38 (lanes 2–5). Coomassie blue staining of purified NURF reconstituted by coexpression of four recombinant subunits (lane 6).
(B) Nucleosome-stimulated ATPase activity. TLC showing hydrolysis of αP32-ATP to αP32-ADP, in reactions (−) or nucleosome core particles (+) containing NURF reconstituted from individual subunits (lanes 3 and 4); NURF reconstituted by coexpression (lanes 5 and 6).
(C) ATPase assay of coexpressed, recombinant NURF, and recombinant ISWI. Lane 2 (65% hydrolysis); lane 6 (29%).
(D) Native gel electrophoresis showing nucleosome mobility. Mononucleosomes (N1-N4) reconstituted on 359 bp DNA (hsp70 position −348 to +11) were reacted with 2 fmole native NURF (lane 1 and 2); recombinant NURF (lanes 3–6); complex of recombinant NURF301 and ISWI (lanes 7 and 8); mixture of recombinant ISWI, NURF55, and NURF38 (lanes 9 and 10); recombinant NURF301 alone (lanes 11 and 12). Protein concentrations were determined by quantitative Western blotting using antibodies to ISWI (lanes 1–10) and NURF301 (lanes 1, 2, 11, and 12). DNA was radiolabeled. Dinucleosomes, di.
(E) Native gel electrophoresis comparing nucleosome sliding of coexpressed, recombinant NURF and recombinant ISWI. Nucleosomes were purified from free DNA on glycerol gradients. The gel was stained with SYBR green I
Molecular Cell 2001 8, DOI: ( /S (01) )

4. Figure 3 Interactions between NURF Subunits
FLAG–ISWI, c-myc-NURF55, and AU1-NURF38 expressed from baculovirus-infected Hi-5 cells were purified and immobilized on antibody beads. Bead pull-down assays were conducted by incubation with in vitro-translated, 35S-labeled NURF subunits. After extensive washes with IB-120 mM KCl, bound proteins were analyzed by SDS-PAGE and autoradiography
Molecular Cell 2001 8, DOI: ( /S (01) )

5. Figure 4 NURF and NURF301 Interact with Nucleosomes
(A) Nucleosomes (359 bp) or duck erythrocyte core particles (60 μl, 10 ng/μl) (lane 1) were incubated with anti-FLAG-NURF beads (20 μl, 200 ng/μl NURF, or 50 ng/μl of ISWI equivalent) or with anti-FLAG beads in nucleosome sliding buffer on ice for 30 min, followed by two to three washes with 500 μl of nucleosome sliding buffer. Bound (lanes 2 and 3) and free (lanes 4 and 5) nucleosomes were analyzed by SDS-PAGE and silver staining.
(B) GST-301 constructs. Seven (I to VII) PCR-generated subfragments of NURF301 were fused to the C terminus of GST. Numbers indicate amino acid positions.
(C) Glutathione-Sepharose beads (Amersham) containing equimolar GST and GST-301 fusion proteins (∼200 ng/μl) were incubated with nucleosomes or core particles and analyzed as in (A). Bottom panel shows a binding reaction using 35S-labeled, in vitro-translated Rpd3 (conditions as in Figure 6C)
Molecular Cell 2001 8, DOI: ( /S (01) )

6. Figure 5 Deletion of HMGA Domain Impairs NURF Functions
(A) SDS-PAGE and SYPRO orange staining of coexpressed recombinant NURF and the corresponding mutant complex lacking residues 1–121 of NURF301 (ΔN301NURF).
(B) SDS-PAGE and silver staining of 359 bp nucleosomes and core particles, free (F) or bound (B), to wild-type NURF or ΔN301NURF. Assay conditions are as in Figure 4A.
(C) Reduced ATPase activity of ΔN301NURF complex. ATPase assays were conducted as indicated at 37°C. Protein concentrations were determined by SYPRO orange staining with a BSA standard.
(D) Nucleosome sliding by WT and mutant NURF complexes. Nucleosomes were purified away from free DNA on glycerol gradients, incubated with NURF complexes as indicated under standard conditions, electrophoresed on a native gel, and stained with SYBR green I
Molecular Cell 2001 8, DOI: ( /S (01) )

7. Figure 6 NURF Interacts with GAGA Factor
(A) SDS-PAGE and Coomassie blue staining of Drosophila proteins that co-IP with NURF. p70 peptide sequences are given.
(B) Specificity of co-IP between NURF and GAGA factor. Immunoprecipitations were conducted with Drosophila embryo nuclear extract (25 μg/μl protein) in a 500 μl reaction using 50 μl Protein A-Sepharose-CL4B beads coated with 5 μg affinity-purified anti-NURF38 or purified chicken IgG, as indicated. Western blot detection was conducted using antibodies against GAGA protein as described (Tsukiyama et al., 1994).
(C) In vitro-translated, 35S-labeled GAGA519 and GAGA581 (3–5 μl in vitro translation mixture diluted in 60 μl of HEGN-150 mM KCl) were incubated on ice for 40 min with anti-FLAG beads or anti-FLAG-NURF beads (∼20 μl beads, ∼200 ng/μl recombinant NURF). Beads were washed four to five times, each with 500 μl of HEGN-150 mM KCl for 5 min. Bound (B) and free (F) proteins were analyzed by SDS-PAGE and autoradiography.
(D) FLAG-NURF binding to 35S-labeled GAGA factor truncations (numbers indicate residues retained); conditions as in (C).
(E) Summary of binding. Values are normalized to full-length GAGA factor, set as 100
Molecular Cell 2001 8, DOI: ( /S (01) )

8. Figure 7 NURF and NURF301 Interact with Transcription Activators
(A) NURF binds to HSF and VP16. GAL4 DNA binding domain (GAL4/1-147), GAL4DBD-HSF, and GAL4DBD-VP16 (Mizuguchi et al., 1997, 2001) labeled with 35S-methionine by in vitro translation were incubated with anti-FLAG or anti-FLAG-NURF beads as in Figure 6C. Bound proteins were analyzed by SDS-PAGE and autoradiography.
(B) GST-301 pull-down assays using equimolar GST-301 fusion proteins as in Figure 4C and 35S-labeled activators as in (A).
(C) ΔN301NURF complex shows reduced binding to GAGA581 but not HSF and VP16. 35S-labeled activators bound (B) and free (F), after incubation with immobilized-NURF complexes as in (A).
(D) Binding of other NURF subunits to activators. 35S-labeled activators were incubated with immobilized-NURF or NURF subunits at room temperature for 40 min and processed as in (A)
Molecular Cell 2001 8, DOI: ( /S (01) )