Tandem Ubiquitin Binding Entity (TUBE) Assay

BLA tissue was homogenized in buffer (10 mM HEPES, 1.5 mM MgCl₂, 10 mM KCl, 0.5 mM DTT, 0.5% IGEPAL, 0.02% SDS, 70 mM NEM, 1 μl/ml protease inhibitor cocktail, and 1 μl/ml phosphatase inhibitor cocktail). The concentration of NEM used has been shown to preferentially preserve linear polyubiquitinated proteins during tissue lysis (Emmerich and Cohen, 2015). Then a linear polyubiquitin-selective tandem ubiquitin binding entity (TUBE) assay was performed. MagnaLink streptavidin magnetic beads (#M-1003-010; Vector Laboratories, Burlingame, CA, USA) were aliquoted (100 μl), washed thoroughly with Wash Buffer (100 mM Tris-HCL, 150 mM NaCl, 5 mM EDTA, 0.08% NP-40) and 4 μl of M1-specific TUBE (#UM-0306-0050; Life Sensors, Malvern, PA) was added, followed by incubation for 2 h on rotator at 4°C. Beads were then washed and a 500 μl mixture of protein (300 μg for all samples and both sexes), protease inhibitor (10 μg/μl), and Wash Buffer was added to each tube, followed by incubation for 2 h on rotator at 4°C. Samples were then washed twice and incubated at 96°C for 5 min at 800 rmp in 1X sample buffer. After cooling at room temperature, the supernatant was collected and stored at −80°C for mass spectrometry analysis.