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. 2014 Apr 6;289(20):13810–13820. doi: 10.1074/jbc.M113.543322

FIGURE 4.

FIGURE 4.

Sox9 promotes the secretion of cartilage matrix proteins through the activation of BBF2H7-Sec23a signaling. A, Western blotting of Sox9, BBF2H7 and Sec23a in WT and Bbf2h7−/− primary cultured chondrocytes infected with adenoviruses expressing Sox9. Note that in Bbf2h7−/− primary cultured chondrocytes, the expression of BBF2H7 and Sec23a was inhibited despite the expression of Sox9. B, quantitative analysis of Sec23a expression in A. Data are shown as ratios of Sec23a and β-actin (n = 3). C, Western blotting of Sox9 and Sec23a in primary cultured chondrocytes transfected with Sox9-targeting siRNA. D, quantitative analysis of Sox9 and Sec23a expression in C. Data are shown as ratios of each protein and β-actin (n = 3). E, Western blotting of Col2 and Col11 using cell lysates and culture medium collected from primary cultured chondrocytes infected with adenoviruses expressing Sox9. Note that the amounts of Col2 and Col11 proteins secreted into the extracellular space were increased upon expression of Sox9. Col2, Col11, or Ihh in the culture medium (right panels) was immunoprecipitated using anti-Col2, anti-Col11, or anti-Ihh antibodies, respectively. F, quantitative analysis of Col2 and Col11 proteins in E. Data are shown as ratios of each protein and β-actin (cell lysates) or Ihh (culture medium) (n = 4). G, Western blotting of Col2 and Col11 using cell lysates and culture medium collected from WT and Bbf2h7−/− primary cultured chondrocytes infected with adenoviruses expressing Sox9 and BBF2H7 N terminus. Col2, Col11 or Ihh in the culture medium (right panels) was immunoprecipitated using anti-Col2, anti-Col11, or anti-Ihh antibodies, respectively. The secretions of Col2 and Col11 into the extracellular space were inhibited in Bbf2h7−/− primary cultured chondrocytes expressing Sox9 alone. H, quantitative analysis of Col2 and Col11 proteins in G. Data are shown as ratios of each protein and β-actin (cell lysates) or Ihh (culture medium) (n = 3). Mock indicates empty vector. Values represent mean ± S.D. *, p < 0.05; **, p < 0.01; ***, p < 0.001.