Figure 4: CagA binds and depletes endogenous GSK-3.

(a) Following the infection with 61090, time-course protein abundances of CagA and GSK-3β in Triton X-100-soluble or insoluble fractions were determined by immunoblot analysis in cells. Note the decrease in GSK-3β protein abundance of soluble fraction in a time-dependent manner. Tubulin was used as the loading control. (b) The wt or mutant CagA expression vectors were co-transfected with a HA-tagged GSK-3β expression vector in 293 cells for a 48 h period. The transition of GSK-3β into insoluble fractions was detected by immunoblot analysis against anti-HA antibody. (c) The endogenous GSK-3 kinase activity in AGS and MKN28 cells was measured. The samples from 0 h, 8 h and 12 h with time-course negative control were subjected to the kinase assay following infection with 60190 or CagA-deleted 60190 (ΔCagA), or with untreated control. The relative kinase activity was determined from triplicate experiments. **P<0.01 compared with control and to ΔCagA treatment by Student’s t-test. Error bars indicate s.d. (d) The AGS and MKN28 cells were infected with control or 60190 or CagA-deleted 60190 (ΔCagA) for 2 h. The GSK-3–CagA complexes were isolated in immunoprecipitates recovered from the lysates following pull-down with anti-GSK-3β monoclonal antibody and immunoblot with anti-CagA or polyclonal GSK-3β antibody. Mouse IgG was used for negative control (–) of anti-GSK-3β antibody. (e) AGS cells were transfected with HA-tagged GSK-3β expression vector in combination with control (upper panels) or 60190 infections (lower panels). The cells were stained with anti-CagA (green) and anti-HA to localize GSK-3β (red), and DAPI to visualize nuclei (blue) by confocal microscopy. White arrows in the merged image indicate co-localized membranous GSK-3 and CagA. Scale bar, 20 μm. (f) AGS cells were infected with negative control (left panels) or 60190 (right panels) and endogenous Snail was detected by immunofluorescence (green). Scale bar, 20 μm.