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. 2011 Oct 15;22(20):3812–3825. doi: 10.1091/mbc.E11-04-0328

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© 2011 He et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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FIGURE 8: — SGK3-mediated activation of NHE3 is dependent on PI3K and PDK1. (A) PS120/NHE3V/HA-SGK3 cells were pretreated with 1 μM RU or 20 μM LY prior to treatment with 1 μM Dex or carrier for 15 min. SGK3 protein was purified by immunoprecipitation, and the kinase activity was determined by TR-FRET. n = 3. (B) PS120/NHE3V/HA-SGK3 cells were stably transfected with sh-PDK1 or pLKO.1, and the expression level of PDK1 was determined by Western blotting using an anti-PDK1 antibody. (C) PS120/NHE3V/HA-SGK3 cells transfected with sh-PDK1 or pLKO.1 were treated with 1 μM Dex or carrier for 15 min. SGK3 protein was purified, and kinase activity was determined. n = 3. (D) NHE3 activities were determined in PS120/NHE3V/HA-SGK3 cells pretreated with RU or LY prior to treatment with Dex. (E) NHE3 activities in PS120/NHE3V/HA-SGK3 cells transfected with sh-PDK1 or pLKO.1 are shown. (A–E) n.s., not significant. *, p < 0.01 and **, p < 0.05, compared with respective controls. n = 6. (F) PS120/NHE3V cells expressing HA-SGK3 were serum-starved and treated with Dex or carrier for 15 min. Colocalization of SGK3 (green) and phospho-PDK1(p-PDK1, red) was determined using anti-HA and anti-p-PDK1 antibodies, respectively. (G) The graph represents Pearson's coefficient of SGK3 and p-PDK1 colocalization from 10 independent fields of cells. *, p < 0.01 compared with the control. (H) Colocalization of GFP-Rab11 (green) and p-PDK1 (red) in PS120/NHE3V cells treated with or without Dex for 15 min is shown. Scale bar: 10 μm. Insets show high magnification of merged signals. (I) Pearson's coefficient of GFP-Rab11 and p-PDK1 colocalization from 10 independent fields of cells. *, p < 0.01 compared with the control.