Figure 4.

FoxO1-induced protein degradation is proteasome dependent and requires new protein synthesis. A, FoxO1–3A-ER cells were differentiated (Diff) for 2 d in the presence or absence of 1 μm 4-HT with or without 1 μm MG132 as indicated, followed by Western blot analysis of the lysates. B, The cells were differentiated up to 48 h in the presence of 1 μm 4-HT. Some cells were exposed to 5 μm cycloheximide (CHX) at either 0 or 24 h of differentiation as indicated. The cell lysates were analyzed by Western blotting. Note that the S6K1 band displayed decreased mobility in CHX-treated samples, suggesting phosphorylation of this protein induced by CHX, consistent with reported observations (48).