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. 2020 Feb 6;117(8):4199–4210. doi: 10.1073/pnas.1916164117

Fig. 2.

Fig. 2.

Sox10 regulates Neurog2 protein stability. (A) Dorsal view of chicken embryos transfected with tdTomato+Neurog2-GFP (n = 6) or Sox10-tdTomato+Neurog2-GFP (n = 7) at 6, 12, and 24 hpt. (B) Quantification of relative fluorescence intensities (GFP/tdTomato) in embryos transfected with the indicated constructs at each time point. (C) Cross-sections of embryos electroporated with the indicated constructs. Nuclei were counterstained with DAPI. (a′, a″, b′, and b″). The magnified areas are marked with dashed boxes. (a′) The majority of Neurog2-GFP proteins were localized in the nucleus of cells expressing vector control. (a″ and b″) White arrows indicate cells expressing strong nuclear+cytoplasmic Neurog2-GFP. (b′) White solid arrowhead indicates nuclear localization of Neurog2-GFP only. (b′ and b″) Open arrowheads indicate cells expressing weak nuclear+cytoplasmic Neurog2-GFP. (D) Quantification of the number of tdTomato+GFP+ cells in each treatment. (E) Graph showing the percentage of cells with nuclear Neurog2-GFP only, nuclear (strong)+cytoplasmic Neurog2-GFP, and nuclear (weak)+cytoplasmic Neurog2-GFP in the total number of GFP+ cells in embryos treated with the indicated constructs at 24 hpt. (F) Well-transfected chicken embryos with the indicated constructs (n = 10 per treatment) were subjected to immunoprecipitation (IP) with anti-Flag and blotted with anti-Ub and anti-Flag. A total of 20% of the total input was blotted with anti-Sox10 and anti-Flag. Gapdh served as a loading control. (G) Densitometric quantification of the levels of Flag-Neurog2 conjugated to ubiquitin (Ub) in each treatment relative to the control. (H) Cross-sections of embryos electroporated with control morpholino (Ctrl-MO; n = 8) or Sox10-MO (n = 8) at 24 hpt. The magnified areas are marked with dashed boxes. (I) Graph showing ratio of Neurog2+ cells between electroporated and unelectroporated sides of embryos transfected with the indicated constructs. (J) Immunofluorescence for GFP and Neurog2 on transverse sections of Sox10N/+ and Sox10N/N mouse embryos at E9.5. (K) Quantification of the number of Neurog2+ migratory NCCs of Sox10N/+ (n = 5) and Sox10N/N mutants (n = 5) at E9.5. Error bars ± SEM (**P < 0.01, ***P < 0.001). Hc, heavy chains. (Scale bars: embryos, 20 μm; sections, 50 μm.)