Fig. 4

AKT directly phosphorylates Sox9 at serine 181. a The consensus AKT target sequence is provided aligned to bona fide AKT substrates (GSK-3b, FOXO3A, GABRB2, and SOX2) and the putative consensus sequence within SOX9 at serine 181. Within the consensus sequence X represents any amino acid, the phosphorylated serine (S) or threonine (T) is in red and ϕ is any hydrophobic residue. A recombinant kinase assay was performed using 200 ng of recombinant active GST-AKT and GST-Sox9 as substrate (b) or a GST-Sox9 S181A mutant (c). d Phosphorylation of Sox9 at serine 181 was assessed by Western blot analysis in SLK^fl/fl and SLK^-/- NDL cells treated with DMSO or MK-2206 for 72 hours. e Luciferase assays were performed in the presence or absence of MK-2206 using the responsive −6904/−5995 element of the Sox10 promoter. Raw light units from the luciferase constructs were normalized to Renilla for each technical sample. Data is represented as the mean luciferase activity from three independent biological replicates +/− SEM. ns: no statistical difference, *p < 0.05