Figure 5.

Analysis of polyubiquitin chains linked to transforming growth factor β–activated kinase 1 (TAK‐1) in injured cartilage compared to that stimulated with interleukin‐1 (IL‐1) or tumor necrosis factor (TNF). Porcine metacarpophalangeal joints were processed and cartilage lysates were prepared as described in Materials and Methods. A, TAK‐1 was immunoprecipitated (IP) and analyzed by Western blotting (WB) for K⁶³‐linked ubiquitin (UB‐K⁶³) or for TAK‐1 phosphorylated at T187. B, TAK‐1 was immunoprecipitated from injured cartilage, cartilage stimulated with IL‐1 for 10 minutes, and cartilage stimulated with TNF for 10 minutes. Immunocomplexes bound to beads were washed and then divided into 9 tubes and used in ubiquitin chain restriction enzyme analysis as described in Materials and Methods. A different deubiquitinase (DUB) was used in each tube as indicated. After incubation, supernatants were electrophoresed and gels were stained with silver to detect released ubiquitin chains (primary analysis). Asterisks indicate the position of recombinant deubiquitinase enzymes. +VE = positive. C, TAK‐1 was immunoprecipitated from injured cartilage and then treated with AMSH or otubain 1 (OUTB‐1) deubiquitinases as in B (primary analysis). Supernatants were analyzed by silver staining, and beads were mixed with 1× sample buffer and analyzed by Western blotting with anti‐ubiquitin antibody (secondary analysis). Representative results from 3 independent experiments are shown. Color figure can be viewed in the online issue, which is available at http://onlinelibrary.wiley.com/journal/doi/10.1002/art.39965/abstract.