Figure 5.

14-3-3 proteins are required for Akt-mediated nuclear accumulation of LKB1. (A). HEK293 cells were transfected with scramble control or 14-3-3ζ-specific siRNA (si14-3-3ζ). After 48 hours, the level of 14-3-3ζ was detected by Western blotting using an anti-14-3-3ζ antibody. (B). The effects of 14-3-3ζ knocking down on sub-cellular distribution of LKB1 were evaluated in the nuclear (Nuc) and cytosolic (Cyt) fractions of HEK293 cells stably overexpressing Flag-tagged wild type LKB1. Western blotting was performed using an anti-Flag antibody. (C). HEK293 cells stably overexpressing Flag-tagged wild type LKB1 were transfected with or without difopein, and subjected to co-immunoprecipitation using anti-Flag M2 agarose beads. 14-3-3 proteins associated with LKB1 were detected by Western blotting using an anti -pan-14-3-3 antibody. (D). The nuclear and cytosolic fractions of Flag-tagged wild type LKB1 in cells treated with or without difopein were detected using an anti-Flag antibody. The relative ratios of the protein bands in Western blotting results were calculated and presented as the means ± SEM from at least three independent experiments. * P <0.05 vs corresponding controls.