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. Author manuscript; available in PMC: 2014 May 23.
Published in final edited form as: Cell Rep. 2014 Apr 24;7(3):681–688. doi: 10.1016/j.celrep.2014.03.048

Figure 4. Botch deglycinates Notch1 and Notch1 E1669 is required for Botch to block Notch1 signaling.

Figure 4

(A) Thin layer chromatography of γ-glutamyl-Glycine cleavage by either recombinant GGCT or Botch, but not Botch-E115A. (n = 3).

(B) Thin layer chromatography of NECD liberation of glycine by Botch. Four reference amino acids were run along with the cleavage product. Rfx100 for NECD cleavage product is 26. (n = 3).

(C) Immunoblots of Notch1 or Notch1-E1669Q cleavage by furin. Abbreviations: FL, Full Length; ECD, Extracellular domain.

(D) Optical densitometry quantification of a normalized to IgG heavy chain. Values represent the mean ± SEM (n ≥ 3; *** p < 0.001; n.s. p > 0.05; Student’s t-test).

(E) Immunoblot analysis of Notch1 in whole cell lysate and surface expression following overexpression of Botch and Flag-Notch1-GFP or Flag-Notch1-E1669Q-GFP.

(F) Optical densitometry quantification of (E) normalized to β-tubulin. Values represent the mean ± SEM (n = 3; ** p < 0.01; n.s. p > 0.05; one-way ANOVA, post-test: Tukey’s multiple comparison tests).