TrkC-KF associates to the transcription factor Hey1 in the nucleus, and both bind to the chromatin.

<p>(A,B) Expression of TrkC-KF-GFP and Hey1-RFP in N2A cells indicates a partial colocalization in the nucleus, as shown by confocal analysis (A) and by the associated Pearson’s coefficient (B). As controls, TrkC, TrkC-642-825, and Neo-IC (fused to GFP) were also transfected. Data represent mean ± SEM (2 independent experiments). *<i>p</i> < 0.05. <i>t</i> test compared to control (TrkC-GFP). (C) Proximity ligation assay (DuoLink) using an anti-Hey1 antibody (recognizing endogenous Hey1) and an anti-GFP antibody on SHEP cells transfected with TrkC-KF-GFP, TrkC-642-825-GFP, GFP, or TrkC-KF-GFP and an siRNA against Hey1: The protein–protein interactions are visualized by red fluorescent spots (Cy3). (D) Quantification of the proximity ligation assay presented in (C): Data represent mean ± SEM (4 independent fields). **<i>p</i> < 0.01. <i>t</i> test compared with TrkC-KF. (E) Immunoprecipitation of TrkC-KF-GFP and TrkC-642-825-GFP using an anti-GFP antibody in HEK293T transfected cells. Hey1 and HeyL are revealed by an anti-Flag WB. (F) HEK293T cells transfected with TrkC-KF-GFP and Hey1-Flag constructs were fractionated into cytoplasmic, nuclear/DNA-unbound, and nuclear/DNA-bound fractions. Actin and Histone H3 are used as loading controls. “Input” corresponds to construct expression in whole cell lysates. Underlying data can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002912#pbio.2002912.s001" target="_blank">S1 Data</a>. GFP, green fluorescent protein; HEK293T, human embryonic kidney 293 T; IP, immunoprecipitation; N2A, Neruo2a; Neo-IC, intracellular fragment of Neogenin; RFP, red fluorescent protein; siRNA, small interfering RNA; TrkC, tropomyosin receptor kinase C; TrkC-FL, full-length TrkC; TrkC-KF, TrkC killer-fragment; WB, western blot.