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. Author manuscript; available in PMC: 2012 Aug 1.
Published in final edited form as: Biochim Biophys Acta. 2011 May 19;1813(8):1453–1464. doi: 10.1016/j.bbamcr.2011.05.001

Fig. 6. Intact activation sites and ATP binding pocket are required for AKT to bind FoxO3 and enhance its steady-state protein levels.

Fig. 6

(A and B) HEK-293T cells were transfected for 24 hours with HA-FoxO3 (lanes 1–5) or HA-FoxO3 3XA (lanes 6–10) together with GST, GST-AKT (4 or 6 µg DNA), GST-AKT K179M or GST-AKT K179M/2A (K179M/T308A/S473A). Immunoblots showing expression of HA-FoxO3, T32-phosphorylated FoxO3, tubulin and the GST-fusion proteins in total cell extracts are presented in A and the recoveries of HA-FoxO3 and GST-fusion proteins following GST-pull-down are presented in B.

(C) HEK-293T cells were transfected for 24 hours with HA-FoxO3 together with GST (lane 1), GST-AKT (lane 2), GST-AKT 2A (T308A/S473A, lane 3) or GST-AKT K179M (lane 4). Immunoblots showing expression of HA-FoxO3 and the GST-fusion proteins in total cell extracts are presented in the left panel and the recoveries of HA-FoxO3 and GST-fusion proteins following GST-pull-down are presented in the right panel.

(D) HEK-293 cells were transfected with HA-FoxO3 together with GST control (lanes 4–6) or GST-AKT (lanes 1–3) and 24 hours after transfection the media was replaced with fresh complete media (lanes 1 and 4) or media lacking serum supplemented with vehicle (lanes 3 and 6 or with 20µM LY294002 (lanes 2 and 5). Immunoblots showing recovery of HA-FoxO3 following GST-pull-down are presented in the right panel. Also shown are GST-fusion protein recoveries and expression of HA-FoxO and pFoxO3 in total cell extracts (left two panels).