Research Paper Volume 4, Issue 7 pp 462—479

Figure 4. FoxO3 is methylated at K271 in cells. (A) Scheme for generation of K271me1 antibody. A branched mono-methylated 11 amino acid peptide surrounding FoxO3 K271 was used as the epitope. (B) The K271me1 antibody is specific to FoxO3 that has been methylated at K271 in in vitro methylation assays. In vitro methylation assays were conducted with cold SAM and analyzed by western-blot with the K271me1 antibody [33]. Coomassie staining was used to show equal levels of FoxO3 and Set9 were used in each condition (bottom). ◁: FoxO3, ◀: FoxO3 degradation product. (C) The K271me1 antibody is specific to FoxO3 K271 methylation in cells. Flag-FoxO3 and Flag-Set9 (WT or H297A methyltransferase-deficient mutant) were co-expressed in 293T cells, and FoxO3 methylation was analyzed by western-blot with the K271me1 antibody. (D) Endogenous Set9 methylates ectopically expressed FoxO3. The methylated form of FoxO3 was immunoprecipitated from a 293T cell line with a stable knock-down of Set9 using the K271me1 antibody and the IP eluates were analyzed by western-blot with the Flag antibody. (E) Endogenous FoxO3 is methylated by overexpressed Set9. 293T cells were transfected with Flag-Set9, and FoxO3 was immunoprecipitated with an antibody to total FoxO3. The IP eluates were analyzed by western-blot with the K271me1 antibody. (F) Endogenous FoxO3 is methylated by endogenous Set9. Methylated FoxO3 was immunoprecipitated from the stable Set9 shRNA cell line used in (D) using the K271me1 antibody. The IP eluate was analyzed by western-blot with an antibody to total FoxO3. ◁: FoxO3, *: non-specific band.