Anti ubiquitin ab

Whole-cell lysates (30 μg protein/lane) were loaded in 10% SDS-PAGE gels and transferred to nitrocellulose membranes. Immunoblotting was carried out using Abs to Itch (BD), IL-1β (Santa Cruz), MMP13 (Abcam), β-Actin (Sigma). Bands were visualized using Clarity Western ECL substrate (BioRad). Cells were lysed in lysis buffer, including NEM, at a concentration of 5 mM and subjected to IP^{13 (link)}. Proteins (500 μg) were diluted in 100 μl lysis buffer containing 1 μg of anti-Itch Ab followed by prewashed EZview Red Protein G Affinity Gel beads (Millipore). The bound antigens were eluted and fractionated by 8% SDS-PAGE gel and incubated with anti-ubiquitin Ab (Santa Cruz).

Ten-week-old female C57BL/6J mice were treated with Vehicle, Btz or BP-Btz and sacrificed at different time points. Legs containing femora and tibiae were dissected free of soft tissue and bone marrow cells were flushed out. Bones samples of epiphysis and metaphysis were frozen in liquid nitrogen and immediately mashed in a mortar to extract proteins using Ubiquitination Lysis Buffer (1X RIPA, EMD 20-188; 1mM DTT; 1mM PMSF; 5mM NEM; 1μg/mL ubiquitin aldehyde). Levels of total ubiquitinated (Ub-) proteins were determined by Western blot analysis using an anti-Ubiquitin Ab (Santa Cruz, cat.#sc-88017). β-Actin was used as a loading control. The relative total Ub-protein levels (fold change over Vehicle.) were calculated.

Btz and Veh were i.a. injected into the right or the left joint, respectively. Mice were sacrificed 4 hours later. Synovial tissues and cortical bone of femur were harvested after removal of BM. Proteins were extracted using Ubiquitination Lysis Buffer. Total ubiquitinated proteins were determined by Western blot using an anti-Ubiquitin Ab (Santa Cruz). β-actin was used as a loading control.