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. 2018 Aug 24;46(21):11214–11228. doi: 10.1093/nar/gky756

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© The Author(s) 2018. 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 Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

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Figure 2. — FUBP1 protein interacts with RUNX1 in hematopoietic lineages. (A) Number of peptides from RUNX1 (49 kDa), CBFB (22 kDa) and FUBP1 (68 kDa) proteins identified by RIME (rapid immunoprecipitation mass spectrometry of endogenous proteins) with RUNX1 or immunoglobulin G (IgG) control in pre-B lymphoblastic Nalm6 cells. The lysates were incubated with 10 μg of anti-RUNX1 (ab23980, Abcam), 5 μg of anti-RUNX1 (HPA004176, Sigma Aldrich) or with 10 μg of normal anti-IgG (sc2027, Santa Cruz Biotechnology). (B) Co-immunoprecipitation (IP) using anti-Flag antibody (M2 clone) in HEK293 cells expressing Halotag-RUNX1 and/or Flag-FUBP1. Western blots were performed with RUNX1 and FUBP1 antibodies. Similar levels of IgG heavy chains in both IP-Flag lanes were used as loading control. (Ci) Representative images of a Proximity Ligation Assay (PLA) in Nalm6 cells. Four pairs of antibodies were used as indicated (references of the antibodies are listed in the Supplementary Table S1). Nuclei were stained by DAPI and appeared in blue. Colocalization of proteins, visualized by PLA dots, appeared in green. (Cii and Ciii) Quantitation of protein co-localization per nucleus–visualized by PLA dots- in Nalm6 cells, presented with the mean values ± S.D. The value of the mean is indicated at the top of each scatter dot plot. Quantitation of PLA dots was performed by an automatic counting with ImageJ as published by Debaize et al. (42). Positive controls (named total RUNX1 or total FUBP1, where primary antibodies against two different epitopes of RUNX1 or FUBP1 were used) and negative controls (either anti-RUNX1 alone or anti-FUBP1 alone) were included. Here, the positive threshold value represented by the dotted line (set at two S.D over the background signal) is 4.3 dots. One representative experiment of at least two independent experiments is shown. (Ciii) CBP and P300 are co-activators that are well known to interact with RUNX1. (D) Quantitation of protein colocalization per nucleus (visualized by PLA dots) in human mononuclear cells from bone marrow of three pre-B acute lymphoblastic leukemia patients, presented with the mean values. The value of the mean is indicated at the top of each scatter dot plot. Here, the positive threshold value represented by the dotted line is 1.7 dots.