The phenotype of DAPT-treated lungs is not associated with inhibition
ofγ-secretase cleavage of E-cadherin. A, Western
blot analysis of NICD, E-cadherin (C36 antibody), β-catenin, and panactin
in 48 h cultured lungs in which Notch cleavage was blocked by DAPT (50
μm). There is no difference in the pattern of staining or
intensity of the full-length E-cadherin (E-Cad/FL), E-cadherin fragment
potentially cleaved by γ-secretase (E-Cad/CTF2), or β-catenin. The
graph represents densitometry measurements of Western blots (mean
± S.E.; values normalized by panactin; n = 3/group).
B, whole mount immunostaining of E-cadherin (green) and
F-actin (red) in E12 lung shows strongest E-cadherin signals in
distal buds. C, WMISH of Titf1 in lungs cultured in control,
E-cadherin blocking antibody (antiE; 100 μg/ml), and DAPT (50
μm)-containing media (48 h). AntiE inhibits branching
(arrowhead) but does not result in the enlargement of peripheral buds
(yellow arrow) or ectopic budding (circled area)
characteristic of DAPT-treated lungs. n = 3/condition.