Figure 5
WNK1 silencing reduced FOXO4 phosphorylation at different sites from those phosphorylated by Akt. (A) Immunoblots showing that FOXO4 phosphorylation at T32, S197, or S262, which promotes FOXO4 nuclear exclusion and inactivates the protein, was not decreased by WNK1 silencing in C2C12 cells. (B) Domain structure of human FOXO4 protein. (C) Phos-tag SDS-PAGE revealed that the abundance of upper smear bands representing phosphorylated full-length FOXO4 [1–505] was decreased in WNK1-silenced HEK 293 T cells compared with that in control cells (Fig. S3B,C). The abundance of phosphorylated bands of FOXO4 [1–312] was unchanged in WNK1-silenced cells, whereas that of FOXO4 [313–505] was decreased. FH/DBD, forkhead box/DNA-binding domain; NLS, nuclear localization sequence, NLS; NES, nuclear export sequence; WNK1, with-no-lysine (K); FOXO4, Forkhead box protein O4. Full-length blots are presented in Supplementary Fig. 11.
