Figure 5. Stabilization of β-catenin by... | Rockefeller University Press

$Figure 5. Stabilization of β-catenin by SOXC proteins. (A and B) β-Catenin and SOX9 protein levels in 10T1/2 cells transfected with GFP and empty (−) or 3FLAG-SOX11 (+) expression plasmids. After 24 h, untransfected and transfected cells were sorted by flow cytometry and whole-cell extracts (A) or membranous/cytoplasmic (mbr/cyt.) and nuclear extracts (B) were made. Western blotting was performed with FLAG antibodies for SOX11 and specific antibodies for other proteins. Numbers indicate protein levels measured in representative blots and normalized with β-actin (A) or GAPDH and HDAC1 (B) levels. Thin vertical white lines added in blot pictures indicate that the order of lanes was rearranged for presentation clarity. (C) TOP-Flash reporter activity in 10T1/2 cells transfected with empty (−) or FLAG-SOXC expression plasmids. Extracts were made after 24 h. Reporter activities are presented for one representative experiment as means of triplicates with standard deviation after normalization for transfection efficiency. The Western blot shows the levels of SOXC and nonspecific (NS) proteins recognized by FLAG antibodies. (D, left) qRT-PCR assay of Ctnnb1 RNA level in Ctnnb1fl/fl osteoblasts infected with lacZ or CRE adenovirus for 24 h and with GFP or SOX11 adenovirus for the next 24 h. Data are shown as means of biological triplicates with standard deviation after normalization with Gapdh mRNA level. Please note that the slight increase in Ctnnb1 mRNA level observed in SOX11-compared with GFP-expressing cells was not statistically significant. (right) β-Catenin level in Ctnnb1fl/fl osteoblasts infected with lacZ or CRE adenovirus for 24 h and then transfected with GFP and empty (−) or SOX11 (+) expression plasmids for another 24 h. GFP-positive/lacZ and GFP-positive/CRE cells were sorted by flow cytometry. Representative Western blots are shown for subcellular fractions. Relative protein levels were quantified using GAPDH, HDAC1, and Na+/K+ ATPase as controls. All data were reproduced in at least three distinct experiments. Each panel shows the results of a representative experiment.$

Figure 5.

Stabilization of β-catenin by SOXC proteins. (A and B) β-Catenin and SOX9 protein levels in 10T1/2 cells transfected with GFP and empty (−) or 3FLAG-SOX11 (+) expression plasmids. After 24 h, untransfected and transfected cells were sorted by flow cytometry and whole-cell extracts (A) or membranous/cytoplasmic (mbr/cyt.) and nuclear extracts (B) were made. Western blotting was performed with FLAG antibodies for SOX11 and specific antibodies for other proteins. Numbers indicate protein levels measured in representative blots and normalized with β-actin (A) or GAPDH and HDAC1 (B) levels. Thin vertical white lines added in blot pictures indicate that the order of lanes was rearranged for presentation clarity. (C) TOP-Flash reporter activity in 10T1/2 cells transfected with empty (−) or FLAG-SOXC expression plasmids. Extracts were made after 24 h. Reporter activities are presented for one representative experiment as means of triplicates with standard deviation after normalization for transfection efficiency. The Western blot shows the levels of SOXC and nonspecific (NS) proteins recognized by FLAG antibodies. (D, left) qRT-PCR assay of Ctnnb1 RNA level in Ctnnb1^fl/fl osteoblasts infected with lacZ or CRE adenovirus for 24 h and with GFP or SOX11 adenovirus for the next 24 h. Data are shown as means of biological triplicates with standard deviation after normalization with Gapdh mRNA level. Please note that the slight increase in Ctnnb1 mRNA level observed in SOX11-compared with GFP-expressing cells was not statistically significant. (right) β-Catenin level in Ctnnb1^fl/fl osteoblasts infected with lacZ or CRE adenovirus for 24 h and then transfected with GFP and empty (−) or SOX11 (+) expression plasmids for another 24 h. GFP-positive/lacZ and GFP-positive/CRE cells were sorted by flow cytometry. Representative Western blots are shown for subcellular fractions. Relative protein levels were quantified using GAPDH, HDAC1, and Na⁺/K⁺ ATPase as controls. All data were reproduced in at least three distinct experiments. Each panel shows the results of a representative experiment.

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