Figure 1.
Rapamycin-resistant SGK1 phosphorylation is modulated by mTOR. (A) mpkCCD cells were grown on collagen-coated Transwell polycarbonate membranes and treated with aldosterone and insulin for 4 hours, followed by treatment with inhibitors as shown for 1 hour. Lysates were prepared and analyzed by immunoblot using antibodies against holo-SGK1, phospho-HM, Akt phospho-S473, and phospho-p70S6K, as shown. α-Tubulin is shown as loading control. (B) Phosphatase treatment eliminates mobility-shifted phospho-SGK1 band. mpkCCD cells were grown on Transwell filters and treated with aldosterone and insulin as in A, and whole-cell lysates were treated with λ-phosphatase (λ-PPase) before Western blotting analysis with antibodies, as shown. (C) Immunodetection of HM phosphorylated SGK1 using a commercially available antibody (S422; Santa Cruz Biotechnology). HEK-293 cells were transfected with FLAG-SGK1 or vector control; incubated with insulin for 4 hours; and treated with inhibitors for 1 hour at concentrations of 20 μM for LY, 0.3 μM for PP242, and 0.1 μM for rapamycin. FLAG-SGK1 was enriched by immunoprecipitation with anti-FLAG antibody (see text for details) and immunoblotted as shown.