Figure 5. Immunoblot analysis of Dnmt1 expression in cells expressing mutant or wild-type β-catenin.

(A) Immunoblot analysis of cells expressing wild-type β-catenin (CTNNB1^WT/−-HCT116) and mutant β-catenin (CTNNB1^−/Δ45 -HCT116). Total soluble proteins were extracted and compared in two separate clones for each cell-line, 20ug of total protein was loaded on SDS-PAGE and western blots were performed using α-β-catenin, α-Dnmt1 and α-α-tubulin (loading control). (B) Immunoblot analysis of sub-cellular fractions of cells expressing wild-type β-catenin (CTNNB1^WT/−-HCT116) and mutant β-catenin (CTNNB1^−/Δ45-HCT116) (two clones for each cell type). For each fraction, a total protein of 20ug was loaded on SDS-PAGE and western blots were performed using α-β-catenin and α-Dnmt1. (C) Wnt3A time course stimulation of CTNNB1^WT/−-HCT116 (clone D075) and CTNNB1^−/Δ45-HCT116 cells (clone K058). Cells were collected at 0, 1, 3, 6 and 24 hours post Wnt3A (30ng/ml) stimulation on each cell line followed by total protein extraction. Total protein of 20ug was loaded on SDS-PAGE and western blots were performed using α-β-catenin and α-Dnmt1.