American Society of Hematology

Figure 3

$Figure 3. Recurrent FOXO1 mutations alter phosphoinositide 3-kinase/AKT phosphorylation and protein-protein interactions. (A) Whole cell extracts from DLBCL cells were resolved on 4% to 12% Bis-Tris gels, following by immunoblotting (left) with anti-FOXO1 antibody. Total protein level was assayed by immunoblot using anti–β-tubulin antibody. *Mutant FOXO1 cell lines. The smaller molecular weight band in the OCI-Ly1 and Su-DHL-5 cell lines, hemizygous and heterozygous for the M1 mutation, respectively, indicates a shift in translation initiation to the next methionine. Relative FOXO1 protein expression (normalized to β-tubulin) is displayed in increasing expression on the right. Where possible, the expression from the individual alleles was determined; (wt/mt) indicates an unknown proportion of expression from the wild-type and mutant protein. (B) HEK-293 cells were transiently transfected with constructs encoding FOXO1 (wild type or mutants) C-terminally tagged with GFP. Twenty-four hours after transfection, cells were serum starved for 24 hours and treated with 10% fetal bovine serum and 100 ng/mL recombinant IGF-1 for 1 hour. Whole cell extracts were resolved on 4% to 12% Bis-Tris gels and immunoblotted with phospho-specific antibodies directed against FOXO1. The total level of tagged protein was assayed by immunoblot using the anti-GFP and anti-FOXO1 antibodies. A reduction in phosphorylation at T24 was observed in the N-terminal mutants but not in the wild type. (C) HEK-293 cells were transiently transfected with constructs encoding FOXO1 (wild type or mutants) C-terminally tagged with GFP. Whole cell extracts were immunoprecipitated with GFP-Trap, resolved on 4% to 12% Bis-Tris gels, followed by immunoblotting with anti-GFP antibody. Blocked magnetic beads were used as a negative control. A loss of interaction with 14-3-3 was observed with the N-terminal mutants but not in the wild type. W, whole cell extract; IP, immunoprecipitation; -‘ve IP, negative control IP. (D) HEK-293 cells were stably transfected with constructs encoding FOXO1 (wild type or mutants) C-terminally tagged with V5. Cells were serum starved for 24 hours and treated with 10% fetal bovine serum and 100 ng/mL recombinant IGF-1 for 1 hour. Biochemical fractionation was performed, and cytoplasmic and nuclear fractions were resolved on 4% to 12% Bis-Tris gels and immunoblotted with anti-FOXO1 antibody. *V5-tagged FOXO1. Note: wt-V5, R21C-V5, and T24A-V5 are the upper bands, and M1L-V5 is the lower band. Purity of the cytoplasmic and nuclear fractions was assayed by immunoblot using anti–β-tubulin and anti-TBP antibodies, respectively. An inability to be exported from the nucleus to the cytoplasm following IGF-1 treatment was observed in our mutants but not for wild-type FOXO1. Cyt, cytoplasmic fraction; Nuc, nuclear fraction; SF, serum-free media.$

Recurrent FOXO1 mutations alter phosphoinositide 3-kinase/AKT phosphorylation and protein-protein interactions. (A) Whole cell extracts from DLBCL cells were resolved on 4% to 12% Bis-Tris gels, following by immunoblotting (left) with anti-FOXO1 antibody. Total protein level was assayed by immunoblot using anti–β-tubulin antibody. *Mutant FOXO1 cell lines. The smaller molecular weight band in the OCI-Ly1 and Su-DHL-5 cell lines, hemizygous and heterozygous for the M1 mutation, respectively, indicates a shift in translation initiation to the next methionine. Relative FOXO1 protein expression (normalized to β-tubulin) is displayed in increasing expression on the right. Where possible, the expression from the individual alleles was determined; (wt/mt) indicates an unknown proportion of expression from the wild-type and mutant protein. (B) HEK-293 cells were transiently transfected with constructs encoding FOXO1 (wild type or mutants) C-terminally tagged with GFP. Twenty-four hours after transfection, cells were serum starved for 24 hours and treated with 10% fetal bovine serum and 100 ng/mL recombinant IGF-1 for 1 hour. Whole cell extracts were resolved on 4% to 12% Bis-Tris gels and immunoblotted with phospho-specific antibodies directed against FOXO1. The total level of tagged protein was assayed by immunoblot using the anti-GFP and anti-FOXO1 antibodies. A reduction in phosphorylation at T24 was observed in the N-terminal mutants but not in the wild type. (C) HEK-293 cells were transiently transfected with constructs encoding FOXO1 (wild type or mutants) C-terminally tagged with GFP. Whole cell extracts were immunoprecipitated with GFP-Trap, resolved on 4% to 12% Bis-Tris gels, followed by immunoblotting with anti-GFP antibody. Blocked magnetic beads were used as a negative control. A loss of interaction with 14-3-3 was observed with the N-terminal mutants but not in the wild type. W, whole cell extract; IP, immunoprecipitation; -‘ve IP, negative control IP. (D) HEK-293 cells were stably transfected with constructs encoding FOXO1 (wild type or mutants) C-terminally tagged with V5. Cells were serum starved for 24 hours and treated with 10% fetal bovine serum and 100 ng/mL recombinant IGF-1 for 1 hour. Biochemical fractionation was performed, and cytoplasmic and nuclear fractions were resolved on 4% to 12% Bis-Tris gels and immunoblotted with anti-FOXO1 antibody. *V5-tagged FOXO1. Note: wt-V5, R21C-V5, and T24A-V5 are the upper bands, and M1L-V5 is the lower band. Purity of the cytoplasmic and nuclear fractions was assayed by immunoblot using anti–β-tubulin and anti-TBP antibodies, respectively. An inability to be exported from the nucleus to the cytoplasm following IGF-1 treatment was observed in our mutants but not for wild-type FOXO1. Cyt, cytoplasmic fraction; Nuc, nuclear fraction; SF, serum-free media.

This Feature Is Available To Subscribers Only