The NICD’s PPxY tyrosine integrates Abl, Su(dx), and Nedd4Lo-mediated regulation of Notch trafficking and signaling at LEs. (A) Left panel: In vitro kinase assay showing that recombinant c-Abl can phosphorylate the NICD, with some activity targeted to the Y2328 residue. Right panel: Western blot showing GST-NICD loading in samples evaluated on left panel. Only the more prevalent degradation products, but not the full-length GST-NICD band shown in the kinase assay panel, were detected with the anti-NICD antibody. (B–E″) Single confocal sections of representative S2 cells showing Notch (indirect immunofluorescence) and Rab7-EYFP. “n” marks the cell nucleus. Transfections shown: (B) Notch^Y2328F, (C) Notch^Y2328F + Abl, (D) Notch^Y2328F + Su(dx), and (E) Notch^Y2328F + Nedd4Lo. Scale bars = 5 µm. (F) Notch signal profiles across LE diameters in manipulations in B–E. Sample size (# of LEs evaluated): Notch (WT, N = 38), Notch^Y2328F (N = 43), Notch^Y2328F + Abl (N = 73), Notch^Y2328F + Su(dx) (N = 38), and Notch^Y2328F + Nedd4Lo (N = 52). (G) Relative Notch activity in manipulations in B–E. A t-student means comparison test was performed, and P values <0.05 (*), <0.01 (**), and <0.001(***) are indicated. Source data are available for this figure: SourceData F6.