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. 2017 Apr 11;6:e24419. doi: 10.7554/eLife.24419

Figure 2. O-GlcNAc on NOTCH1 EGF repeats promotes DLL4-NOTCH1 interactions.

(A) Ala substitution of Thr/Ser in the O-GlcNAc consensus site C5XXG(Y/F)(T/S)GXXC6 in EGF2, 10, 17, and 20 in the NOTCH1Δ4xO-GlcNAc mutant. (B) Cell surface expression of NOTCH1Δ4xO-GlcNAc is comparable to that of wild-type NOTCH1. EOGT-null transfectants expressing Notch1 or Notch1Δ4xO-GlcNAc with or without Eogt were subjected to flow cytometry using 8G10 NOTCH1 antibody. Control transfectants were with (Mock) or without (Cont) primary antibody. Similar results were obtained for wild-type HEK293T cells (not shown). (C) Expression and O-GlcNAcylation of Notch1, Notch1Δ2xO-GlcNAc and Notch1Δ4xO-GlcNAc were analyzed by immunoprecipitation (IP) using NOTCH1 (8G10) antibody, followed by immunoblotting with CTD110.6 O-GlcNAc or NOTCH1 antibodies. (D) HEK293T transfectants expressing NOTCH1, NOTCH1Δ2xO-GlcNAc or NOTCH1Δ4xO-GlcNAc were immunostained for O-GlcNAc (CTD110.6 mAb; red) or NOTCH1 (8G10 mAb; green). Merged images include DAPI (blue) staining. Note that CTD110.6 mAb binding is markedly decreased in the NOTCH1Δ4xO-GlcNAc mutant. (E) Quantification of the number of DLL4- or JAG1-coated Dynabeads bound to Notch1 versus Notch1Δ4xO-GlcNAc transfected cells (marked with GFP), in the presence and absence of Eogt. Data are mean ± S.D from three independent experiments. Each experiment analyzed 50 cells. *p<0.05; **p<0.01; ***p<0.001 (Welch's t test).

DOI: http://dx.doi.org/10.7554/eLife.24419.006

Figure 2—source data 1. Raw data for Figure 2E.
DOI: http://dx.doi.org/10.7554/eLife.24419.007
elife-24419-fig2-data1.xlsx (41KB, xlsx)
DOI: 10.7554/eLife.24419.007

Figure 2.

Figure 2—figure supplement 1. NOTCH1 O-GlcNAc site mutants.

Figure 2—figure supplement 1.

(A) Schematic of the EGF repeats of mammalian NOTCH1 identifying the potential O-GlcNAc consensus site, C5XXG(Y/F)(T/S)GXXC6, on EGF2, 10, 11, 14, 15, 17, 20, 23, 26, 27 and 29. These consensus sites are partly conserved in zebrafish and Drosophila. Recent studies of Drosophila Notch identified EGF4, 11, 12, 14 and 20 as major O-GlcNAcylated sites (Harvey et al., 2016). (B) Schematic of EGF repeats of mouse Notch1Δ2xO-GlcNAc and Notch1Δ4xO-GlcNAc.
DOI: http://dx.doi.org/10.7554/eLife.24419.008