Fig 1: Notch internalization is triggered by ligand binding and vesicle acidification is necessary for receptor activation.(A) Representative frames from live-cell imaging of LysoTracker-stained Ctrl#2-NSCs starting after addition of DLL1-pHrodo at time point 0. LysoTracker- and pHrodo single-positive vesicular structures are indicated by red and green arrows, respectively, double-positive structures with yellow arrows. (B) Representative kymographs from confocal live-cell imaging of Ctrl#2-NSCs treated with LysoTracker and DLL1-pHrodo. LysoTracker- and pHrodo single-positive vesicular structures are indicated by red and green arrows, respectively, and double-positive structures are indicated by yellow arrows. (C) Immunostaining for Notch1, LAMP1, and 6xHis after incubation of Ctrl#2-NSCs with DLL1-6xHis for 30 min. Arrows indicate puncta colocalizing for all three proteins. (D to F) Western blot against cleaved N1ICD, total Notch1, and actin and respective quantifications of Ctrl#1-NSCs treated with 0.1% (v/v) DMSO, 20 µM DAPT, 100 nM BafA, or 200 µM Leu for 2 hours (n = 4, one-way ANOVA with Bonferroni’s multiple comparison test, means ± SEM). (G) Fold changes of gene expression of Notch downstream targets HES1, HES5, and HEY1 after treatment of Ctrl#2-NSC with 0.1% (v/v) DMSO, 20 µM DAPT, 100 nM BafA, or 200 µM Leu for 2 hours (n = 3, one-way ANOVA with Bonferroni’s multiple comparison test, means ± SEM). Scale bars, 20 µm (A and C), 5 µm [zoomed in (C)], and 10 µm (B). Time scale, hh:minmin:ss (A). *P < 0.05. See also fig. S2.