Figure 6. SOXC proteins inhibit... | Rockefeller University Press

$Figure 6. SOXC proteins inhibit β-catenin degradation in the destruction complex and repress Sox9 expression. (A) Effect of SOX4 on the activity of wild-type and CK1/GSK3-insensitive (stabilized) β-catenin. HEK293 cells were transfected with TOP-Flash and control reporters, 100 ng empty (−), or 3FLAG-SOX4 expression plasmid, and 0–200 ng of β-catenin expression plasmid. Normalized TOP-Flash activities are presented as mean values with standard deviation of triplicate cultures in a representative experiment. (B) Effect of SOX4 on the levels of wild-type and stabilized β-catenin. Representative blots of membrane/cytoplasm and nuclear extracts from HEK293 cells transfected with 200 ng of wild-type and 20 ng of stabilized β-catenin–FLAG plasmids, respectively. Levels of β-catenin were normalized to HDAC1 and GAPDH levels in nucleus and membrane/cytoplasm (mbr/cyt.) fractions, respectively. *, P < 0.05; n = 3 experimental replicates. (C) Detection of proteins binding to Axin in 10T1/2 cells infected with lacZ or 3FLAG-SOX11 adenovirus for 24 h. Cell lysates (inputs) were immunoprecipitated with nonimmune (IgG-IP) or Axin1 antibodies (Axin-IP). Western blots are shown for a representative experiment. Equal amounts of cell lysates were used for control (lacZ) and tester (SOX11) samples, as shown with GAPDH levels in inputs. SOX11 overexpression was reproducibly found to increase the level of Axin1. (D) β-Catenin–independent repression of Sox9 by SOX11. (left) Ctnnb1fl/fl osteoblasts were infected with lacZ or CRE adenovirus for 44 h and with GFP or SOX11 adenovirus for the next 24 h. Representative blots are shown for whole-cell extracts. (right) qRT-PCR assay in replicate samples. Ctnnb1 and Sox9 RNA levels, normalized to those of Gapdh, are shown as means of triplicates with standard deviation. (E) Levels of total and phosphorylated β-catenin in limb bud extracts from E12.5 SoxCPrx1Cre, Sox9Prx1Cre, and respective control littermates. β-Catenin is phosphorylated at residue S45 by CK1 and at residues S33, S37, and T41 by GSK3. Representative blots are shown. (F) Levels of total and phosphorylated β-catenin in whole extracts of 10T1/2 cells infected with lacZ or 3FLAG-SOX11 adenovirus for 24 h. Representative blots are shown. (G) Levels of SOX9 and total and phosphorylated β-catenin in whole extracts of Sox9fl/fl limb bud cells infected with lacZ or CRE adenovirus for 16 h and then with GFP or 3FLAG-SOX11 adenovirus for 24 h. Representative blots are shown. Levels of total and phosphorylated β-catenin assessed in triplicates were normalized to β-actin levels. *, P < 0.05 between lacZ/GFP controls and other samples; #, P < 0.05 between lacZ/SOX11 and Cre/SOX11 samples. Note that Sox9 depletion has opposite effects on the level of β-catenin phosphorylated by GSK3 in limb bud cells in vivo and in vitro (E and G). The differences may reflect differential effects of Sox9 deletion on cell fate or behavior in the two experimental models. All data were replicated three or more times. Each panel shows typical results.$

Figure 6.

SOXC proteins inhibit β-catenin degradation in the destruction complex and repress Sox9 expression. (A) Effect of SOX4 on the activity of wild-type and CK1/GSK3-insensitive (stabilized) β-catenin. HEK293 cells were transfected with TOP-Flash and control reporters, 100 ng empty (−), or 3FLAG-SOX4 expression plasmid, and 0–200 ng of β-catenin expression plasmid. Normalized TOP-Flash activities are presented as mean values with standard deviation of triplicate cultures in a representative experiment. (B) Effect of SOX4 on the levels of wild-type and stabilized β-catenin. Representative blots of membrane/cytoplasm and nuclear extracts from HEK293 cells transfected with 200 ng of wild-type and 20 ng of stabilized β-catenin–FLAG plasmids, respectively. Levels of β-catenin were normalized to HDAC1 and GAPDH levels in nucleus and membrane/cytoplasm (mbr/cyt.) fractions, respectively. *, P < 0.05; n = 3 experimental replicates. (C) Detection of proteins binding to Axin in 10T1/2 cells infected with lacZ or 3FLAG-SOX11 adenovirus for 24 h. Cell lysates (inputs) were immunoprecipitated with nonimmune (IgG-IP) or Axin1 antibodies (Axin-IP). Western blots are shown for a representative experiment. Equal amounts of cell lysates were used for control (lacZ) and tester (SOX11) samples, as shown with GAPDH levels in inputs. SOX11 overexpression was reproducibly found to increase the level of Axin1. (D) β-Catenin–independent repression of Sox9 by SOX11. (left) Ctnnb1^fl/fl osteoblasts were infected with lacZ or CRE adenovirus for 44 h and with GFP or SOX11 adenovirus for the next 24 h. Representative blots are shown for whole-cell extracts. (right) qRT-PCR assay in replicate samples. Ctnnb1 and Sox9 RNA levels, normalized to those of Gapdh, are shown as means of triplicates with standard deviation. (E) Levels of total and phosphorylated β-catenin in limb bud extracts from E12.5 SoxC^Prx1Cre, Sox9^Prx1Cre, and respective control littermates. β-Catenin is phosphorylated at residue S45 by CK1 and at residues S33, S37, and T41 by GSK3. Representative blots are shown. (F) Levels of total and phosphorylated β-catenin in whole extracts of 10T1/2 cells infected with lacZ or 3FLAG-SOX11 adenovirus for 24 h. Representative blots are shown. (G) Levels of SOX9 and total and phosphorylated β-catenin in whole extracts of Sox9^fl/fl limb bud cells infected with lacZ or CRE adenovirus for 16 h and then with GFP or 3FLAG-SOX11 adenovirus for 24 h. Representative blots are shown. Levels of total and phosphorylated β-catenin assessed in triplicates were normalized to β-actin levels. *, P < 0.05 between lacZ/GFP controls and other samples; ^#, P < 0.05 between lacZ/SOX11 and Cre/SOX11 samples. Note that Sox9 depletion has opposite effects on the level of β-catenin phosphorylated by GSK3 in limb bud cells in vivo and in vitro (E and G). The differences may reflect differential effects of Sox9 deletion on cell fate or behavior in the two experimental models. All data were replicated three or more times. Each panel shows typical results.

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