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. 2012 Apr 10;4(2):175–186.

Figure 3.

Figure 3

The interaction between 14-3-3 and LKB1 is dependent on the phosphorylation of LKB1 at Ser334. (A). GST-14-3-3ζ bound to Glutathione Sepharose 4B beads was incubated with the cell lysates of HEK293 cells treated with DMSO (vehicle control) or 10 μM Akti-1/2. The endogenous LKB1 associated with GST-14-3-3ζ was detected by Western blotting using an anti-LKB1 antibody. (B). HEK293 cells overexpressing Flag-tagged wild type LKB1 were treated with or without 10 μM Akti-1/2, followed by co-immunoprecipitation using anti-Flag M2 agarose beads. 14-3-3 proteins associated with Flag-tagged LKB1 were detected by Western blotting using an anti-pan-14-3-3 antibody. (C). HEK293 cells overexpressing wild type LKB1 (WT), S334A, or S334D were incubated with GST-14-3-3ζ-bound Glutathione Sepharose 4B beads, and the immunoprecipitated proteins were detected by Western blotting using an anti-Flag antibody. (D). HEK293 cells overexpressing wild type LKB1 (WT), S334A or S334D were subjected to co-immunoprecipitation using anti-Flag M2 agarose beads, and endogenous 14-3-3 proteins associated with Flag-LKB1 were detected by Western blotting using an anti-pan-14-3-3 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.