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. 2009 Jan 16;284(3):1612–1619. doi: 10.1074/jbc.M806981200

FIGURE 4.

FIGURE 4.

Effect of PI3K p110α siRNA on VacA-induced Akt phosphorylation and effects of LY294002 and silencing of the PI3K gene on VacA-induced phosphorylation at Ser-9 of GSK3β. A, AZ-521 cells were transiently transfected with siRNA for PI3K p110α or negative siRNA as a control as described under “Experimental Procedures.” The transfected cells were incubated with 120 nm VacA at 37 °C for up to 60 min. After incubation, cell lysates were subjected to SDS-PAGE in 10% gels, and then transferred to PVDF membranes for Western blot analysis with the antibodies against phospho-Ser-473 Akt, Akt, and PI3K p110α. Quantification of phospho-Ser-473 Akt obtained with VacA (filled bars) and iVacA (open bars) was determined by densitometry scan analysis. Data are means ± S.E. of values from triplicate experiments, with an n = 3 per experiment. Statistical significance: *, p < 0.05. B, AZ-521 cells were pretreated with 50 μm LY294002 for 30 min before incubation with 120 nm VacA or iVacA at 37 °C. After incubation with VacA or iVacA for indicated times, the cell lysates were applied to SDS-PAGE in 10% gels. The separated proteins were transferred to PVDF membranes, and subjected to Western blot analysis with anti-phospho-Ser-9 GSK3β and GSK3β antibodies. Quantification of phospho-Ser-9 GSK3β was determined by densitometry for VacA (filled bars) and iVacA (open bars). Data are means ± S.E. of values from triplicate experiments, with an n = 3 per experiment. Statistical significance: *, p < 0.05. C, AZ-521 cells were transiently transfected with 200 pmol of siRNA for PI3K p110α or negative siRNA as negative control as described under “Experimental Procedures.” The transfected cells were incubated with 120 nm VacA or iVacA at 37 °C for indicated times. After incubation, the cell lysates were subjected to SDS-PAGE in 10% gels, and then transferred to PVDF membranes for Western blot analysis with anti-phospho-Ser-9 GSK3β, GSK3β, and PI3K p110α antibodies. Quantification of phospho-Ser-9 GSK3β obtained with VacA (filled bars) and iVacA (open bars) was determined by densitometry. Data are means ± S.E. of values from triplicate experiments, with an n = 3 per experiment. Statistical significance: *, p < 0.05.