Fig 1: CK1a, not CK1d or CK1e, regulates FOXO4 protein abundance in a mutant RAS-specific manner. (a) CK1a depletion increases FOXO4 protein abundance in a mutant K-RAS-dependent manner. HCT-116 K-RAS isogenic cells were transfected with 100 nM siCtrl or siCK1a (#13 or #14) for 48 h prior to SDS–PAGE/western blot (WB) analysis using the indicated antibodies (n=3). (b) CK1a is the only non-membrane bound CK1 isoform that phosphorylates FOXO4 in vivo. Each of the indicated CK1 isoforms in HCT-116 cells was depleted with two independent siRNAs (48 h) prior to SDS–PAGE/WB analysis using the indicated antibodies (n=3). (c) Inhibition of CK1a, not CK1d/e, increases FOXO4 protein abundance. Cells were treated with the indicated compounds for 16 h prior to SDS–PAGE/WB analysis using the indicated antibodies (n=3). (d) CK1a inhibition increases FOXO4 protein abundance in a time-dependent manner. Cells were incubated with the indicated compound over 10 h prior to SDS–PAGE/WB analysis using the indicated antibodies (n=3). Eg5 serve as the loading control for a–d. (e) Depletion of CK1a by RNAi or (f) inhibition of CK1a by D4476 increases nuclear FOXO4 protein abundance. Cells were (e) transfected with the indicated siRNAs (100 nM; 48 h treatment) or (f) treated with the indicated drugs for 4 h prior to subcellular fractionation and SDS–PAGE/WB analysis using the indicated antibodies (n=3). Lamin B and ß-tubulin serve as the loading control for nuclear and cytosolic fractions, respectively.