Research Paper Volume 4, Issue 7 pp 462—479

Figure 5. The methylated form of FoxO3 has similar localization properties to FoxO3. (A) Immunofluorescence in NIH 3T3 cells expressing FoxO3-GFP (green), using the K271me1 antibody (red). Nuclei were stained with DAPI (blue). White bar: 10 mm. (B) Quantification of FoxO3-GFP localization in U87 cells. Cells were co-transfected with FoxO3-GFP and empty vector (pcDNA), Set9 (WT or H297A), or a constitutively active version of Akt as a positive control (Akt CA). Localization was scored as ‘nuclear’, ‘cytoplasmic’ or ‘ubiquitous’. The data represent the mean and SEM from 3 independent experiments. In each experiment, at least 200 cells per condition were scored. *** p<0.001, one way ANOVA, Bonferroni post-test (C) Chromatin fractionation of 293T cells co-transfected with Flag-FoxO3 and Flag-Set9. Cells were grown in media with 10% FBS (Normal Serum) or in media without serum in the presence of 20 μM LY294002 (Serum Starvation+LY294002) and then fractionated. A subset of the fractions was treated with micrococcal nuclease to digest the chromatin. Presence of chromatin-bound proteins in fractions was assessed by western-blot with antibodies to the core nucleosome histone H2B (H2B). S2: detergent lysis, S3: hypotonic lysis fraction, Ch: chromatin fraction, Nuc: supernatant from nuclease digest of chromatin.