FOXO3a activity inhibition by IKK is dependant on S644 of FOXO3a. (A) MV4-11 cells were infected with a lentivirus expressing either the FOXO3a-GFP protein, the FOXO3aS644A-GFP mutant protein or only the GFP protein and incubated 48 hours in 10% FCS MEM. Two days after lentiviral infection, the GFP localization was investigated as described in Figure 1C. (B) The percentage of cells with nuclear GFP was quantified by an average of 100 GFP-positive cells. Results of the quantification obtained from 5 independent experiments in MV4-11 cells are presented. The statistical significance was calculated versus the control condition (MV4-11 cells expressing the FOXO3a-GFP fusion protein) by Student t test, and vertical bars indicate SDs. (C) Aliquots of 105 cells MV4-11 cells were infected with a lentivirus expressing either the FOXO3a-GFP protein, the FOXO3aS644A-GFP mutant protein, or only the GFP protein and incubated 48 hours in 10% FCS MEM. During the last hour of incubation, cells were treated or not with IC87114. Protein extracts were then performed and analyzed by Western blot.
