Fig 2: Characterization of recombinant ADAMTS-4_v1. A, Blot of anti–FLAG M2 monoclonal antibody (mAb) immunoprecipitates from Triton X-100 extracts of untransfected HEK 293 cells and ADAMTS4_v1-pCEP4–transfected HEK 293 cells, following sodium dodecyl sulfate–polyacrylamide gel electrophoresis and Coomassie brilliant blue R250 staining. The band marked with an asterisk yielded an N-terminal amino acid sequence of ASPLPRE…, whereas no sequence was obtained from the band marked with a double asterisk. B–D, Triplicate Western blots of anti–FLAG M2 mAb immunoprecipitates from Triton X-100 cell extracts and conditioned media obtained from untransfected, ADAMTS4.1-pCEP4–transfected, or ADAMTS4_v1-pCEP4–transfected HEK 293 cells, probed with anti–FLAG M2 mAb (B), a rabbit antiserum (ab28285) to the carboxyl-terminus of ADAMTS-4 (C), or a rabbit antiserum (ab39201) to the pro domain of ADAMTS-4 (D). E, Western blot of anti–FLAG M2 mAb immunoprecipitates from a Triton X-100 extract of an equal number of Tris buffered saline–washed ADAMTS4_v1-pCEP4–transfected HEK 293 cells (Triton X-100 extract), a Triton X-100 extract of the pellet after high salt extraction (high salt pellet), and a 1M NaCl, 50 mM Tris HCl, pH 7.4, extract of ADAMTS4_v1-pCEP4–transfected HEK 293 cells (high salt extract). F, Western blot probed with anti–FLAG M2 mAb using a plasma membrane protein extraction kit to prepare cytosol, total membranes, and plasma membranes from ADAMTS4_v1-pCEP4–transfected HEK 293 cells prior to anti–FLAG M2 mAb immunoprecipitation.

Fig 3: Proteolysis of ADAMTS-4_v1. A, Western blots probed with anti–FLAG M2 monoclonal antibody (mAb) or affinity-purified polyclonal goat antibodies to human ADAMTS-4 from purified ADAMTS-4_v1 that had been stored at -80°C during the course of the experiment (lane 0) or had been incubated with 2 units of furin at 37°C for 1, 2, or 3 hours (lanes 1, 2, and 3, respectively). B, Western blots probed with affinity-purified AltTS4.2, a rabbit antibody raised against a synthetic peptide representing a unique sequence in the C-terminus of ADAMTS-4_v1, or with affinity-purified goat antibodies to human ADAMTS-4 from purified ADAMTS-4_v1 that had been stored at -80°C during the course of the experiment (lane 0) or had been incubated at 37°C in the presence of 10 mM CaCl2 for 1, 3, or 6 hours (lanes 1, 3, and 6, respectively). C, Western blot probed with anti–FLAG M2 mAb from purified ADAMTS-4_v1 that had been stored at -80°C during the course of the experiment (-80°C) or had been incubated for 6 hours at 37°C in the presence of 10 mM CaCl2 (control) or 10 mM CaCl2 plus 24 nM tissue inhibitor of metalloproteinases 1 (TIMP-1), 23 nM TIMP-2, or 5 mM EDTA.

Fig 4: Aggrecanase substrate activity of ADAMTS-4_v1, as assessed by SensoLyte 520 aggrecanase 1 assay. A, Aggrecanase 1 activity of an anti–FLAG M2 immunoprecipitate from ADAMTS-4_v1 transfected with HEK 293 cells that had been predigested for 1 hour at 37°C with furin (?) or an equal amount of undigested ADAMTS-4_v1 immunoprecipitate (?), as compared to that of the recombinant truncated ADAMTS-4 (•) (supplied in the assay kit). Enzyme activity (expressed as relative fluorescence units [RFU]) was measured every 5 minutes for 1 hour. B, Western blot probed with anti–FLAG M2 monoclonal antibody from anti–FLAG M2 immunoprecipitates from ADAMTS-4_v1–transfected HEK 293 cells used in A. Shown are undigested and furin-digested ADAMTS-4_v1 prior to their use in the assay (preassay) as well as undigested and furin-digested ADAMTS-4_v1 plus recombinant truncated ADAMTS-4 (rADAMTS-4) that was harvested from the assay plate after the 1-hour course of the aggrecanase 1 assay (postassay). C, Duplicate Western blot of B probed with affinity-purified goat anti-human ADAMTS-4 antibodies (BAF4307).

Fig 5: Characterization of antisera raised against the ADAMTS-4_v1 synthetic peptides. A and B, Western blots of Triton X-100 extracts of HEK 293 cells transfected with ADAMTS4_v1-pCEP4 (lane 1), ADAMTS4.1-pCEP4 (lane 2), or left untransfected (lane 3) and probed with the AltTS4.1 serum (A) or the AltTS4.2 serum (B). C, Western blot of a Triton X-100 extract of HEK 293 cells transfected with ADAMTS4_v1-pCEP4 and probed with affinity-purified AltTS4.2 antibodies (lane 1) or with affinity-purified AltTS4.2 antibodies that had been preincubated with 5 µg/ml of the AltTS4.2 peptide (lane 2). D and E, Duplicate Western blots of anti-FLAG immunoprecipitates from Triton X-100 cell extracts (lanes 1, 3, and 5) and conditioned media (lanes 2, 4, and 6) from untransfected HEK 293 cells (lanes 1 and 2), HEK 293 transfected with ADAMTS4.1-pCEP4 (lanes 3 and 4), or HEK 293 cells transfected with the ADAMTS4_v1-pCEP4 (lanes 5 and 6) and then probed with AltTS4.1 serum (D) or AltTS4.2 serum (E).