Figure 7. SOXC proteins amplify canonical WNT/signaling by inhibiting GSK3-dependent phosphorylation of β-catenin. (A) TOP-Flash reporter activity in HEK293 cells transfected with empty (none) or FLAG-SOX11 expression plasmid and treated with various dilutions of WNT3A medium for the last 6 h of culture. Each dot represents one sample. The equations of linear fits are indicated. (B) β-Catenin level in SoxCfl/fl primary limb bud cells infected with GFP or CRE adenovirus for 24 h. Cells were treated with 20% WNT3A medium for the last 8 h. Representative blots are shown. (C) TOP-Flash reporter activity in 10T1/2 cells transfected for 24 h with empty or 3FLAG-SOX11 expression plasmid. None (−) or DKK1 protein was added 1 h before transfection, and 20% WNT3A medium was added for the last 6 h. Normalized reporter activities are presented as means with standard deviation for triplicates in a typical experiment. Fold increases caused by SOX11 are indicated. *, P < 0.05. (D) β-Catenin level in HEK293 cells transfected for 24 h with β-catenin–FLAG, empty (−), and 3FLAG-SOX4 (+) expression plasmids. DKK1 or solvent (−) was added 1 h before transfection, and 20% WNT3A medium was added for the last 6 h. Cell extracts were subjected to Western blot for β-catenin (FLAG antibody) and β-actin (loading control). Data are shown for a representative experiment. (E) Effect of SOX11 on the level and phosphorylation status of LRP6. HEK293 cells were infected with lacZ (−) or 3FLAG-SOX11 (+) adenovirus for 24 h. WNT3A medium was added for the last 8 h at 0 (−) or 20% (+). Whole-cell extracts were assayed in Western blotting. Data are shown for a representative experiment. (F) Levels of total and GSK3-phosphorylated β-catenin in whole-cell extracts from 10T1/2 cells treated with GFP or 3FLAG-SOX11 adenovirus for 20 h and then with 20% WNT3A medium for the indicated times. Data are shown for a representative experiment. (G) Quantification of protein levels detected in the Western blots shown in F. Total and phosphorylated β-catenin levels were normalized with α-tubulin levels. Each dot represents one sample. Data in each panel are representative of the results of at least three experiments. Thin vertical white lines added in blot pictures indicate that the order of lanes was rearranged for presentation clarity.
Figure 7.

SOXC proteins amplify canonical WNT/signaling by inhibiting GSK3-dependent phosphorylation of β-catenin. (A) TOP-Flash reporter activity in HEK293 cells transfected with empty (none) or FLAG-SOX11 expression plasmid and treated with various dilutions of WNT3A medium for the last 6 h of culture. Each dot represents one sample. The equations of linear fits are indicated. (B) β-Catenin level in SoxCfl/fl primary limb bud cells infected with GFP or CRE adenovirus for 24 h. Cells were treated with 20% WNT3A medium for the last 8 h. Representative blots are shown. (C) TOP-Flash reporter activity in 10T1/2 cells transfected for 24 h with empty or 3FLAG-SOX11 expression plasmid. None (−) or DKK1 protein was added 1 h before transfection, and 20% WNT3A medium was added for the last 6 h. Normalized reporter activities are presented as means with standard deviation for triplicates in a typical experiment. Fold increases caused by SOX11 are indicated. *, P < 0.05. (D) β-Catenin level in HEK293 cells transfected for 24 h with β-catenin–FLAG, empty (−), and 3FLAG-SOX4 (+) expression plasmids. DKK1 or solvent (−) was added 1 h before transfection, and 20% WNT3A medium was added for the last 6 h. Cell extracts were subjected to Western blot for β-catenin (FLAG antibody) and β-actin (loading control). Data are shown for a representative experiment. (E) Effect of SOX11 on the level and phosphorylation status of LRP6. HEK293 cells were infected with lacZ (−) or 3FLAG-SOX11 (+) adenovirus for 24 h. WNT3A medium was added for the last 8 h at 0 (−) or 20% (+). Whole-cell extracts were assayed in Western blotting. Data are shown for a representative experiment. (F) Levels of total and GSK3-phosphorylated β-catenin in whole-cell extracts from 10T1/2 cells treated with GFP or 3FLAG-SOX11 adenovirus for 20 h and then with 20% WNT3A medium for the indicated times. Data are shown for a representative experiment. (G) Quantification of protein levels detected in the Western blots shown in F. Total and phosphorylated β-catenin levels were normalized with α-tubulin levels. Each dot represents one sample. Data in each panel are representative of the results of at least three experiments. Thin vertical white lines added in blot pictures indicate that the order of lanes was rearranged for presentation clarity.

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