Fig. 8.

GC inoculation induces the phosphorylation of EGFR, which is required for GC-induced redistribution of β-catenin.

A and B. Polarized HEC-1-B cells were incubated with or without GC for 4 h and then lysed. Cell lysates were subjected to immunoprecipitation using a phosphotyrosine-specific antibody. Immunoprecipitates and the cell lysates were analysed using SDS-PAGE and Western blot, probing for EGFR and β-tubulin as loading controls. Shown are representative blots of three independent experiments (A). Densitometry analysis was performed to determine fold increase over control (B).

C–H. Polarized HEC-1-B (C–E) and T84 (F–H) cells were untreated or pre-treated with the EGFR kinase inhibitor AG1478 (10 μM) for 2 h then apically incubated with live GC in the absence or presence of the inhibitor or gentamicin killed GC for 6 h. Cells were stained for β-catenin and GC and analysed using confocal microscopy. The β-catenin FIR of membrane to cytoplasm was determined (D and G) as described in Fig. 5. Colocalization of GC microcolonies with β-catenin was analysed in HEC-1-B (E) and T84 (H) using Pearson correlation coefficients and the Zen software. Shown are representative images (C and F) and the average FIR (D and G) and coefficient (± SD) (E and H) of three independent experiments (~ 15 cells per experiment). Scale bar, 10 μm, ***P ≤ 0.001; **P ≤ 0.01.