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. 2022 May 17;221(7):e202110167. doi: 10.1083/jcb.202110167

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Figure 4. — The NRG3 NTF is trafficked via early and recycling endosomes. (A) Representative image of a neuron transfected with V5-tagged NRG3 and GFP-tagged Rab5 and additionally labeled for MAP2 to identify dendrites. Magnified area and line scan densitometry illustrate extensive NTF/Rab5 overlap. (B) By contrast, many NTF+ puncta in live-imaged axons lacked corresponding strong punctate signals for Rab5 (white arrowheads), while conversely strongly Rab5+ puncta generally lacked corresponding NTF signals (blue arrowheads). (C) Quantitative NRG3/Rab5 co-localization analysis in MAP2+ dendrites and SNPH+ axons. Data plotted as Mander’s overlap coefficients, representing the mean ± SEM from three independent experiments (n = 16 ROIs). (D) Reduced NTF/Rab5 co-localization in neurons co-transfected with NRG3-mCherry and GFP-Rab5 and treated for 6 h with endocytosis inhibitors Dynole (DYN; 10 µM) or Pitstop 2 (PTS; 20 µM). (E) Likewise, axonal NTF accumulation is similarly reduced in DYN/PTS-treated neurons transfected with NRG3/V5 and SNPH. Data in D are plotted as Mander’s overlap coefficients and in E as integrated density, and each represents the mean ± SEM from three independent experiments (n = 14–16 ROIs). ****, P < 0.0001; **, P < 0.01 (one-way ANOVA with Tukey’s post-hoc test). Scale bars: A, 20 µm; A (ROI) and B, 5 µm.