Cells were washed twice with ice-cold PBS and lysed in lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 % NP-40, 10 mM N-ethylmaleimide) supplemented fresh with protease inhibitor cocktail. Lysates were incubated at 4˚ C for 15 min and clarified by centrifugation at 20,000 xg at 4˚ C for 15 min. Samples were quantified by BCA Protein Assay Kit and diluted to 1 mg/mL with Dilution Buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, protease inhibitor). 1–1.5 mg of protein lysates were incubated with 50 μl of Ubiquilin 1 Tandem UBA Agarose (BostonBiochem AM-130) overnight at 4˚ C. Samples were then washed 3x in High Salt Wash Buffer (50 mM Tris-HCl pH 7.5, 250 mM NaCl, 0.5 % NP-40) and once with 10 mM Tris-HCl, pH 7.5. Samples were eluted by adding Laemlli buffer and incubating at 65˚ C for 15–20 min.
For DUB digestions, affinity captured material was washed 1x with DUB digestion buffer (50mM Tris-HCl, pH 7.5, 150mM NaCl, 20mM DTT). Liquid was removed and beads were resuspended in 20ml of DUB digestion buffer and 5mg of USP2 catalytic domain (Usp2cc; BostonBiochem E-506) for 1hr with gentle shaking at 30˚ C. Beads were washed once in High Salt Wash Buffer and once with 10 mM Tris-HCl, pH 7.5 prior to eluting affinity captured material by adding Laemmli buffer and incubating at 65 ˚ C for 15min.
For DUB digestions, affinity captured material was washed 1x with DUB digestion buffer (50mM Tris-HCl, pH 7.5, 150mM NaCl, 20mM DTT). Liquid was removed and beads were resuspended in 20ml of DUB digestion buffer and 5mg of USP2 catalytic domain (Usp2cc; BostonBiochem E-506) for 1hr with gentle shaking at 30˚ C. Beads were washed once in High Salt Wash Buffer and once with 10 mM Tris-HCl, pH 7.5 prior to eluting affinity captured material by adding Laemmli buffer and incubating at 65 ˚ C for 15min.