TrkC-KF is translocated to the nucleus by importins.

<p>(A) Schematic representation of TrkC-KF, generated after TrkC cleavage by caspase in D495 and D641, in absence of NT-3. A second fragment, TrkC 642–825, is also generated but has no apoptotic activity. (B) Expression of TrkC-FL, TrkC-KF, TrkC 642–825, and Neo-IC fused to GFP in N2A murine neuroblastoma cells. Nuclei are stained with DAPI. (C) Proximity ligation assay (DuoLink) using a pan-importins antibody and an anti-GFP antibody on untransfected SHEP cells or those transfected with TrkC-KF-GFP or TrkC-642-825-GFP: 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 (at least 2 independent fields). *<i>p</i> < 0.05. <i>t</i> test compared with control (untransfected). (E) SHEP cells transfected with either TrkC-KF or Neo- IC-ΔNES and treated or not with 10 μM of pan-importin inhibitor Ivermectin were fractionated into cytoplasmic (Cytoplasm, marker: GAPDH) and nuclear (Nucleus, marker: histone) fractions. (F) Localization of the 2 putative NLSs in TrkC-KF sequence: Mutations of these NLSs used in experiment (G) are indicated in red. TrkC-KF-ΔNLS1/2 is mutated on both NLS1 and NLS2. (G) SHEP cells transfected with the NLS-mutated constructs of TrkC-KF were fractionated into cytoplasmic (Cytoplasm, marker: GAPDH) and nuclear (Nucleus, marker: histone) fractions. (H) Cell death quantification by trypan blue exclusion after transfection of SHEP cells with TrkC-KF, TrkC-KF-ΔNLS1/2, or Bax. Data represent mean ± SEM (<i>n</i> = 3). *<i>p</i> < 0.05. <i>t</i> test. (I) Quantification of luciferase activity in SHEP cells transfected with DCC-IC, TrkC-KF, or TrkC-492-825 constructs fused to the Gal4 DBD, and the gene coding for luciferase under the UAS-Gal4 control. Data represent mean ± SEM. *<i>p</i> < 0.05. <i>t</i> test compared to control (Gal4-DBD). 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>. Bax, B cell lymphoma 2–associated X; DBD, DNA-binding domain; DCC-IC, deleted in colorectal cancer intracellular domain; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GFP, green fluorescent protein; N2A, Neuro2a; Neo-IC, intracellular fragment of Neogenin; Neo-IC-ΔNES, Neo-IC deleted for its nuclear export sequence; NES, nuclear export sequence; NLS, nuclear localization sequence; ns, nonsignificant; NT-3, neurotrophin-3; TrkC, tropomyosin receptor kinsase C; TrkC-FL, full-length TrkC; TrkC-KF, TrkC killer-fragment; UAS, upstream activating sequence; WB, western blot.