Figure 4.

STRADα, but not STRADβ stabilizes LKB1 protein. (A) Representative Western blot of lysates from wild-type, STRADα KO, STRADβ cKO, or STRADα/β double KO embryonic day 16.5 (E16.5) cortex. Actin is a loading control. (B) Quantification of LKB1 protein levels analyzed by Western blot, normalized to wild-type cortical lysate. N ≥15 cortices from at least three litters of each genotype. There is no significant difference between columns 1 and 3 or columns 2 and 4. ***P <0.0001 using one-way ANOVA with Bonferroni’s multiple comparison test. (C) Western blot of Nestin-cre⁺; STRADα^f+ or STRADα^ff lysates across developmental time. Quantification of LKB1 protein, normalized to postnatal day 0 (P0) STRADα^f+; Nestin-cre⁺. N ≥3 brains of each genotype from three litters for each time-point. (D) Western blot of E16.5 cortical lysates showing STRADα protein is significantly reduced following conditional cortical loss of LKB1. (E and F) Quantification of N ≥4 independent LKB1 stability time-courses in HEK cells transfected with epitope-tagged LKB1 protein following cycloheximide-mediated translation inhibition, with three to six replicates of each condition in each experiment. (E) STRADα-2 significantly increases LKB1 half-life, as does STRADβ-1, to a lesser extent. (F) LKB1ΔNLS is as stable as LKB1 + STRADα-2 and LKB1ΔNLS + STRADα-2 is similar to LKB1ΔNLS + Vector. Error bars represent SEM. Repeated measures ANOVA with Dunnett’s multiple comparison test with LKB1 + empty vector as the control was employed. ***P <0.001, **P <0.005. ANOVA, analysis of variance; KO, knockout; SEM, standard error of the mean.