Effect of EGFR on the subcellular distribution of β-catenin in oral cancer cell lines. Time-dependent effects of EGF or AG1478 on the subcellular distribution of β-catenin in OECM1 (A) and SAS (B) cells. Cancer cells were treated with EGF (100 ng/mL) or AG1478 (20 μM/mL) for the indicated time, and β-catenin was assayed by Western blotting. (C) Immunocytochemical staining of β-catenin in oral cancer cells. EGF treatment of OECM1 cells induced scattering of cancer cells, breakup of cell-cell junctions, and decreased level of membranous β-catenin (upper panels). AG1478 treatment of SAS cells led to close cell-to-cell contact and abundant membranous β-catenin (lower panels). Immunofluorescence for β-catenin (Rhodamine, Red) and the nucleus (Dapi) in cultured cells was performed 24 h following treatment. Cells were permeabilized with 100% methanol, blocked with 1% BSA, and incubated with the antibody for 30 min. A rhodamine conjugated antibody was used as the secondary antibody.