Fig 1: Mutations of S401 in SGK1 turn motif impact the phosphorylation level at pS422. A, S422 phosphorylation level of SGK1 WT and indicated mutants in transfected 293T cells. B, quantification analysis of pS422 signal as the ratio of pS422/total SGK1. Bars indicate the mean ± SD of four to five independent experiments. Statistical analysis by one-way ANOVA and Dunnett’s post hoc test: ∗p < 0.02, ∗∗p < 0.002, ∗∗∗∗p < 0.0001. C, WB of NDRG1 pT346 and NEDD4L pS448 (two substrates of SGK1) phosphorylation level with indicated SGK1 constructs and the presence of AKT1 specific inhibitor AZD5363 (10 µM) applied for 4 h. The inhibitor was added to identify the component of NDRG1 phosphorylation mediated by SGK1 as the site T346 is also phosphorylated by AKT1. D, WB of pS422 and total SGK1 of WT SGK1 and indicated mutants ± 100 nM Cal A for 15 min.