Figure 3. The Dnmt1- β-catenin association is mutually stabilizing.

(A) Knock-out of CTNNB1 leads to reduced abundance of endogenous Dnmt1. Twenty micrograms of total protein lysate from CTNNB1^KO-HCT116 or HCT116 cells were loaded on SDS-PAGE followed by Western blot analyses with anti-Dnmt1, anti-β-catenin, anti-ɣ -catenin, anti-HAUSP, and anti-GAPDH. One step RT-PCR was performed on the total RNA extracted from both HCT116 and CTNNB1^KO HCT116 cells with specific primer pairs for Dnmt1 and GAPDH (as loading control).(B) Knock-out of DNMT1 leads to reduced abundance of endogenous β-catenin. Twenty micrograms of total protein lysate from DNMT1^KO-HCT116or HCT116 cells were loaded on SDS-PAGE followed by Western blot analyses with anti-Dnmt1, anti-β-catenin and anti-GAPDH. One step RT-PCR was performed on the total RNA extracted from both HCT116 and DNMT1^KO-HCT116 cells with specific primer pairs for CTNNB1 and GAPDH (as loading control). M refers to standard protein marker. (C) Dnmt1 protein expression is rescued in CTNNB1^KO-HCT116 cells by addition of β-catenin. β-catenin protein expression level was restored by making transient transfection in CTNNB1^KO-HCT116 cells with plasmid vector containing full length β-catenin. Total protein lysate (20 μg) from HCT116, CTNNB1^KO-HCT116 or CTNNB1^KO-HCT116 with ectopic expressed β-catenin cells were loaded on SDS-PAGE followed by Western blot analyses with anti-Dnmt1, anti-β-catenin, anti-β-catenin (active) and anti-GAPDH (loading control). M refers to standard protein marker. (D) β-catenin expression is not rescued in DNMT1^KO-HCT116 cells by addition of Dnmt1 protein. Transient transfection of DNMT1^KO-HCT116 cells with plasmid vector containing full length Dnmt1 does not restore β-catenin expression. Total protein lysate (20 μg) from HCT116, DNMT1^KO-HCT116 or DNMT1^KO-HCT116 with ectopically expressed Dnmt1 were loaded on SDS-PAGE followed by Western blot analyses with anti-Dnmt1, anti-β-catenin and anti-GAPDH (loading control). M refers to standard protein marker. (E) Degradation profiles for β-catenin and Dnmt1 in HCT116, DNMT1^KO-HCT116 or CTNNB1^KO-HCT116 cells following cycloheximide treatment. Degradation rate constants were quantified by measuring the relative intensity of each protein by quantitative Western blotting at 0, 3, and 6 hours after cycloheximide treatment. The intensity data were fit to a first-order decay function to estimate the degradation rate constant, which then was used to calculate the half-life.