Figure 3: Wwp2 activates Sox9 by affecting its nuclear translocation.

(a) Wwp2 interacted physically with endogenous Sox9 as demonstrated by IP assay using ATDC5 cells. Myc-tagged Wwp2 was transfected into ATDC5 cells and total cell lysates were immunoprecipitated 48 h after transfection using rabbit IgG (negative control) or an anti-Sox9 antibody, followed by immunoblotting with an anti-myc antibody. (b) Schematic images of Sox9 deletion mutations. Sox9 contains a high-mobility group domain (HMG, residues 104–182); a proline, glutamine and alanine domain (PQA, residues 339–379); and a proline, glutamine and serine-rich domain (PQS-rich, residues 402–509). (c) Schematic images of Wwp2 deletion mutations. Wwp2 contains a calcium-dependent membrane targeting domain (C2, residues 19–115), four tryptophan-based WW domains (WW, residues 300–477) and a carboxy terminus domain that is homologous to that of E6-AP (HECT, residues 525–870). (d) Co-immunoprecipitation assays between myc-tagged Wwp2 and Flag-tagged deletion mutations of Sox9. GFP expression plasmid was used as a negative control. IB, immunoblotting; In, input. (e) Co-immunoprecipitation assays between HA-tagged Sox9 and V5-tagged deletion mutations of Wwp2. GFP expression plasmid was used as a negative control. (f) Immunohistochemical analysis of Sox9 and Wwp2 in wild-type mouse limbs at E16.5 (DAB staining). Pz, proliferative zone; Pre-Hz, prehypertrophic zone; Hz, hypertrophic zone. Scale bar, 100 μm. (g) Nuclear translocation of Wwp2 in the presence or absence of exogenously overexpressed Sox9 in C3H10T1/2 cells. The nuclear translocation of Wwp2 was associated with Sox9. Scale bar, 20 μm. (h) Luciferase reporter assay using the p89/4×48 Col2a1 reporter plasmid or Col11a2 reporter plasmid in C3H10T1/2 cells. The reporter activities increased in a Wwp2 dose-dependent manner (mean ± s.d.; n=5).