FIG. 9.

ADAMTS-7 associates with and digests GEP. (A) ADAMTS-7 binds to GEP in yeast. Yeast two-hybrid assay to test the interaction of proteins fused to the VP16 AD and proteins fused to the Gal4 DNA binding domain. Each pair of plasmids, as indicated, encoding proteins fused to VP16 (below the line) in the vector pPC86 (i.e., pPC86-c-jun, pPC86-GEP, and pPC86-Rb) and those encoding proteins fused to Gal4 (above the line) in the vector pDBleu (i.e., pDB-c-fos, pDB-ADAMTS-7, and pDB-lamin) were cotransfected into yeast strain MAV203. Yeast transformants were selected on SD-Leu-Trp plates and tested for β-galactosidase activity. The known interaction between c-Jun and c-Fos was used as a positive control, whereas the lack of interaction between Rb and lamin was used as a negative control. (B) ADAMTS-7 binds to GEP in chondrocytes (Co-IP assay). Cell extracts prepared from human chondrocytes were incubated with anti-GEP antibodies, anti-ADAMTS-7 antibodies, or control IgG. The immunoprecipitated protein complex and cell extracts (lane 1, which provides a positive control) were examined by Western blotting with either anti-GEP (top) or anti-ADAMTS-7 (bottom) antibodies. (C) The catalytic domain of ADAMTS-7 digests GEP in a dose-dependent manner. GEP was incubated with various amounts of catalytic domain of ADAMTS-7 (TS7-CD), as indicated; the cleaved products were separated by nonreduced SDS-PAGE, and intact GEP and fragments were detected with polyclonal anti-GEP antibodies. The intact GEP (arrow) and processed fragments are indicated. (D) Intact ADAMTS-7 cleaves GEP. GEP was incubated with either the control (CTR) (lane 3) or ADAMTS-7-conditioned medium (lane 4) for 4 h. The processed products as well as the control and ADAMTS-7 media were detected with anti-GEP antibodies. The intact GEP (arrow) and processed fragments are indicated.