Fig. 4.

FOXO3B is phosphorylated by Akt on Thr117 but does not shuttle from the cytoplasm to the nucleus upon Akt inhibition. A. Western blot of LHCNM2 myoblasts overexpressing FOXO3B treated with DMSO (vehicle) and 200 nM insulin or 2 μM MK-2206 and 200 nM insulin for one hour. Phospho-Thr117 of FOXO3B was detected using an antibody raised against Thr24/32 of FOXO1/3 (Cell Signaling #9464). α-Tubulin is used as the loading control. B. Immunofluorescence of inducible FOXO3B overexpression in LHCNM2 myoblasts. +Dox cells were treated with 0.1 ug/ml Dox for 48 hours and then -/+ Dox cells were treated with DMSO and 200 nM insulin or 2 μM MK-2206 and 200 nM insulin for one hour. Blue is DAPI and green is staining with the FOXO3A antibody (Cell Signaling #2497) at a 1:1000 dilution. The white scale bar is 25 μm. C. A LHCNM2 myoblast line containing a Dox-inducible overexpression construct of wild-type FOXO3A. The cells were treated and stained as in Figure 4B. The white scale bar is 25 μm.