Figure 7: Phosphorylation of LKB1 is essential to rescue the delay in myelination.

(a) Illustration of the retroviral construct containing the serine-431–alanine LKB1 phosphorylation mutant. (b) SC-DRG cocultures were established from the SC-specific LKB1 knockout mice. SCs were infected with the WT or S431A LKB1 retrovirus and immunostained for LKB1 (red). Arrows illustrate the localization of LKB1 to the SC–axon interface. Scale bar=10 μm. (c) SCs infected with the WT or S431A LKB1 retrovirus were immunostained for phospho-Ser-431 LKB1 (red). Arrow illustrates the localization of phospho-ser-431 LKB1 to the SC–axon interface. (d) SCs infected with the WT or S431A LKB1 retrovirus were immunostained for Par-3 (red). Arrow illustrates the localization of Par-3 to the SC–axon interface. Scale bar=10 μm. (e) SCs infected with the WT or S431A LKB1 retrovirus were extracted and analysed with western blot analysis. Samples were probed with antibodies to phospho-ser-431 of LKB1, LKB1, Oct-6 and the myelin protein P0. β-actin serves as a loading control. (f) Quantification of the western blots was accomplished by measuring the relative densitometry. The asterisks represent significance based on Student’s t-test as compared with the WT cultures (*P<0.05) or the WT-LKB1 rescue (**P<0.01). (g) Illustration of the retroviral construct containing the serine-431-aspartic acid LKB1 phosphomimetic. (h–k) SCs infected with the WT, S431A or S431D LKB1 retrovirus were induced to myelinate for 10 days. The cocultures were stained with MBP and the percent of myelin internodes formed by the infected SCs was quantified (h). The error bars represent s.d. and *P<0.05. (i,k) Arrows indicate transduced cells (green) forming myelin (MBP, red). (i) Scale bar=50 μm. (l) Illustration of the co-transduction of the retroviral constructs containing the DN-PKA and the S431D LKB1 phosphomimetic. (m–o) SCs co-transduced with the DN-PKA (green) and WT (m, red) or S431D LKB1 (n,o, red) retrovirus were induced to myelinate for 10 days. The cocultures were stained for MBP. Scale bar=50 μm. (o) Co-transduced SCs (yellow) were not observed to express MBP at anytime. All experiments were performed in triplicate and representative images are displayed.