Figure 4.

mSin1/Arg-83 is required for SGK1 but not Akt phosphorylation.A, Western blot analysis of FLAG-immunoprecipitates (IPs) and whole-cell extracts (WCEs) derived from mSin1 KO HEK293T cells cotransfected with Myc-tagged Rictor, SpyCatcher003-tagged mTOR, HA-tagged mLST8, HA-tagged mSin1 (WT, R81A/R82A/R83A triple mutant and R83A single mutant), and FLAG-tagged SGK1. Cells were transfected and then serum starved overnight and stimulated with 100 nM insulin for 60 min. Whole cell extracts were prepared and subjected to immunoprecipitation with anti-FLAG antibody followed by immunoblotting (IB) as shown and further described in “Experimental procedures”. Blots were probed with antibodies against phosphorylated SGK1 S422 (Fig. 4A-source data 1), total SGK1 (Fig. 4A-source data 2), phosphorylated Akt (Fig. 4A-source data 3), total Akt (Fig. 4A-source data 4), and α-tubulin (Fig. 4A-source data 5), respectively. B, quantification of band intensities from the Western Blot data in (A), presented as column graph of mean ± SD from three independent experiments. ∗∗p < 0.01; ∗∗∗∗p < 0.0001 by one-way ANOVA. NS, not significant. mTOR, mechanistic target of rapamycin.