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. 2014 Apr 19;42(10):6232–6242. doi: 10.1093/nar/gku274

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© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 3. — Mapping the FOXK2–BAP1 interaction domains. (A) Schematic representation of full-length FOXK2 and the GST fusions to the C-terminal (amino acids 1–218) and N-terminal (amino acids 189–660) regions of FOXK2. The positions of the forkhead associated (FHA) and forkhead domains (FOX) are shaded in grey. (B) GST pulldown assays using bacterially expressed GST-tagged FOXK2(1–218) and FOXK2(189–660) and in vitro translated BAP1. Arrows mark the positions of full-length GST–FOXK2 fusion proteins. A total of 10% input is shown. (C) Co-immunoprecipitation experiments to analyse interactions between FOXK2(1–218) and BAP1. HEK293T cells were transfected with the indicated plasmids encoding FOXK2(1–218) fused with the Gal4 DNA binding domain and Flag-tagged BAP1, followed by immunoprecipitation (IP) with anti-Flag antibody and immunoblotting (IB) with either an anti-Flag or an anti-Gal4 antibody. ERK2 is a loading control. The asterisk marks a non-specific signal. (D and E) GST pulldown assay using either GST, or wild-type (WT) or FHA mutant (R58A) versions of the GST–FOXK2(1–218) fusion protein and total cell extracts from U2OS cells. Interacting BAP1, HCFC1, SIN3A and input GST fusion proteins were revealed by IB. Arrows mark the positions of full-length GST–FOXK2 fusion proteins. A total of 3% cell lysate input is shown. Ethidium bromide was added to the GST pulldown reactions where indicated. (F) Co-immunoprecipitation experiments using FOXK2 antibodies for IP from U2OS cells. Co-precipitated endogenous BAP1 was detected by IB. Where indicated, the final co-IP was treated with λ phosphatase. (G) Schematic representation of full-length BAP1 and the indicated N- and C-terminal truncation mutants. The locations of the ubiquitin carboxyl-terminal hydrolase (UCH) domain and the C-terminal domain (CTD) are shown. (H) Co-immunoprecipitation analysis of FOXK2 interactions with BAP1 deletion mutants. HEK293T cells were transfected with the indicated plasmids encoding FOXK2(1–218) fused to the Gal4 DNA binding domain and Flag-tagged full-length or truncated mutants of BAP1, followed by IP with anti-Flag antibody and IB with the anti-Gal4, anti-HCFC1 and anti-Flag antibodies. The asterisk marks a non-specific signal.