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. 2013 Jan 15;126(2):484–496. doi: 10.1242/jcs.113035

Fig. 4.

Fig. 4.

The hypoglycosylated E-cadherin mutant, V13, inhibits DPAGT1 transcription. (A) Immunoblot comparison of mobilities of E-cadherin and V13 with E-cadherin from sparse and dense cells. (B) Immunoblot comparing exogenous FLAG-tagged E-cadherins, in cells transfected with either wild-type E-cadherin (E-cad) or its hypoglycosylated mutant (V13) (***P<0.001). (C) Quantitative PCR of DPAGT1 transcript levels in untransfected control (C), E-cad and V13 cells. (D) Immunoblot of GPT protein levels in control (C), E-cad and V13 cells. (E) Comparison of DPAGT1 transcript decay rates in untransfected cells and cells transfected with either E-cad or V13. Total RNAs were isolated from cells grown in the presence of 5 µg/ml actinomycin D for 0, 4, 6, 8, 12 and 24 hours. Results are from three independent experiments. (F) ChIP analyses of β-catenin, γ-catenin and Tcf at the DPAGT1 promoter in E-cad and V13 cells after normalization to the IgG control (***P<0.001). (G) Luciferase reporter activity from the FOP-DPAGT1 vector in E-cad and V13 cells (***P<0.001). (H) Luciferase reporter activity from the TOP-Flash vector in E-cad and V13 cells (**P<0.005). (I) Luciferase reporter activity from the TOP-Flash vector in E-cad cells stimulated with either conditioned medium alone (CM) or from cells overexpressing either Wnt3a or Wnt5a (***P<0.001). (J) Luciferase reporter activity from the TOP-Flash vector in V13 cells stimulated with either conditioned medium alone (CM) or from cells overexpressing either Wnt3a or Wnt5a (***P<0.001).