Figure 4.

FOXO3a induces GSK3-dependent phosphorylation and proteasomal degradation of c-Myc. (a) DL23 cells were induced with 4-OHT or solvent for 24 h. In all, 25 μM MG132 was added for the final 1, 4 or 5 h, as indicated. c-Myc protein levels were determined by immunoblotting. β-Actin is shown as a loading control. (b) DL23 cells were treated with 4-OHT or solvent for 15 min before addition of 2 μg/ml cycloheximide (CHX). Cells were lysed after 15, 30, 45, 60 or 90 min of CHX treatment. c-Myc protein levels were determined by immunoblotting. β-Actin is shown as a loading control. (c) Quantitation of c-Myc protein levels in the presence of CHX in DL23 cells treated with 4-OHT or solvent from three independent experiments. Data are shown as mean±S.E.M. The symbol ‘^*' indicates statistical significance, as determined by Student's t-test (P<0.05, n=3). (d) DL23 cells were treated with 4-OHT or solvent for 24 h. In all, 25 μM MG132 was added for the final 1, 3 or 5 h, as indicated. Levels of phosphorylated and total c-Myc were determined by immunoblotting. β-Tubulin is shown as a loading control. (e) DL23 cells were treated with 4-OHT or solvent for 24 h in the presence of DMSO or 5 μM of the GSK3 inhibitor SB216763. In all, 25 μM MG132 was added for the final 1, 2.5 or 5 h, as indicated. Levels of GSK3α/β, β-catenin and phosphorylated and total c-Myc were determined by immunoblotting. β-Actin is shown as a loading control