Fig. 7.

β-arrestin1-mediated CXCR4 cell-surface modulation by Notch3 in human leukemic cells. a Left: western blot analysis of Notch3 protein. Right: flow-cytometry of CXCR4 cell-surface expression in scrambled (CTRL) versus specific Notch3 silenced (shNotch3) TALL-1 cells. b Transwell migration assay of CTRL and shNotch3 cells in response to SDF-1 (5–50 ng/ml) (****p < 0.0001; ***p < 0.001, Student’s t-test). c Immunoblot assay of whole-cell extract (WCE) of CTRL and shNotch3 cells (left), and thymocytes from WT (WT₁,₂) and N3-ICtg (N3-ICtg₁,₂) mice (right). Phosphorylated (p-β-arrestin1) and unphosphorylated (β-arrestin1) β-arrestin1 protein levels. d Flow-cytometry analysis of CXCR4 expression of Hek293 transiently transfected with N3-IC (0.7 μg) either in the presence (1.4 μg) of β-arrestin1-wt (wt) or of the mutant S412D (β-arrestin1-S412D). Right panel CXCR4 MFI of at least five experiments (*p < 0.05, Student’s t-test). e Fractionated extract (Nucleus, Cytoplasm) of CTRL and shNotch3 TALL-1 cells and from DP/CD8⁺ thymocytes of WT and N3-ICtg 6–8-week-old mice. See also Figure S4 and S6. f Optical densitometry of β-arrestin1 protein in WT (n = 3) and N3-ICtg (n = 3) thymocytes (**p < 0.01, Student’s t-test). Densitometry was performed on scanned immunoblot images using ImageJ