Skip to main content
. 2012 May 10;7(5):e37111. doi: 10.1371/journal.pone.0037111

Figure 6. SGK1 phosphorylates NCT on Ser437, which facilitates degradation of NCT.

Figure 6

(A) HEK293 cells were transfected with expression vectors encoding for 2 µg of V5-NCT, 4 µg of Flag-SGK1-CA, or Flag-SGK1-DN. After 48 hours of transfection, the cell lysates were subjected to immunoprecipitation with anti-V5 antibody. The immunoprecipitates were immunoblotted with anti-phospho Ser/Thr antibody. (B) HEK293 cells were transfected with expression vectors encoding for 2 µg of V5- NCT, then treated with 1 µM dexamethasone for 24 hours. After 48 hours of transfection the cell lysates were subjected to immunoprecipitation with anti-V5 antibody. The immunoprecipitates were immunoblotted with anti-phospho Ser/Thr antibody. (C) MEF cells from SGK1+/+ and SGK1−/− mice were treated with 1 µM dexamethasone for 24 hours. The cell lysates were subjected to immunoprecipitation with an anti-NCT antibody, and the immunoprecipitates were immunoblotted with anti-phospho Ser/Thr antibody. (D) HEK293 cells were transfected with expression vectors encoding for 2 µg of V5-NCT (WT, S437A), 6 µg of Flag-SGK1-CA. After 48 hours of transfection, the cell lysates were subjected to immunoprecipitation with anti-V5 antibody. The immunoprecipitates were immunoblotted with anti-phospho Ser/Thr antibody. (E) HEK293 cells were transfected for 48 hours with expression vectors encoding for the indicated combinations of 2 µg of V5-NCT, 2 µg of V5-NCT (S437A), 2 µg of APP-Myc, and 4 µg of Flag-SGK1-CA. Cell lysates were subjected to immunoprecipitation with an anti-Myc antibody (9E10), and the immunoprecipitates were immunoblotted with an anti-V5 antibody. (F) HEK293 cells were transfected for 48 hours with expression vectors encoding for the indicated combinations of 2 µg of V5-NCT, 2 µg of Flag-SGK1 CA, 2 µg of Flag-SGK1 DN, 2 µg of HA-PS1, 2 µg of HA-Pen2, and 2 µg of HA-APH-1. Cell lysates were subjected to immunoprecipitation with an anti-V5 antibody, and the immunoprecipitates were immunoblotted with an anti-HA antibody. (A–F) The cell lysates were also subjected to immunoblotting analysis with the indicated antibodies. These results represent one of three independent experiments. IB, Immunoblot. IP, Immunoprecipitation.