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Fig. 5. Co-localization and physical interaction of Brpf1 and Moz.
(A-D) Brpf1 co-localizes with Moz. Immunofluorescent staining of
interphase HEK 293 cells after co-transfection with the indicated versions of
GFP-Brpf1 (left panels; green) and FLAG-Moz (middle panels; red),
counterstained with DAPI (for DNA; blue); merged images are shown in
right-hand panels. Full-length Brpf1 co-localizes with wild-type Moz (A) and
with HAT-negative Moz-G675E (B) in a punctate pattern on interphase nuclei.
Co-localization is abolished when Brpf1 is N-terminally truncated (C).
N-terminal fragment of Brpf1 co-localizes with Moz, but displays a more
diffuse distribution (D). (E,F) Schematic structures and
co-localization/immunoprecipitation properties of full-length Brpf1,
full-length Moz (E), and the various truncations used (F). (G-I) Brpf1
physically associates with Moz. (G) Co-IP of full-length Brpf1 and wild-type
or HAT-negative Moz(G657E) from co-transfected cells with anti-FLAG (Moz)
antibody (left) or anti-HA (Brpf1) antibody (right), analyzed in western blots
(upper panels) with the specified antibodies, or assayed for HAT activity on
core histones (lower panels). (H) Co-IP of full-length FLAG-Moz and various
GFP-Brpf1 deletion constructs with anti-FLAG or anti-GFP antibodies, followed
by analysis of complex formation (upper panel) and control for Brpf1
expression levels (lower panel) via anti-GFP western blotting. (I) Co-IP of
full-length Moz or C-terminally truncated MozN and various HA-tagged versions
of the N-terminal fragments of Brpf1 using anti-FLAG antibody, analyzed in
anti-HA western blots. Lower panel shows input control. Brpf1 aa 1-245
fragments that have histidine or cysteine mutations in the zinc-finger domain
can still co-precipitate with Moz (lanes 5, 6), whereas the aa 1-149 fragment
with an intact zinc finger cannot (lane 4). Scale bars: 5 µm in B; 2.5
µm in D.
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