Sc 390585

For Western blot analyses, control and USP22-depleted HCT116 cells were lysed in radioimmunoprecipitation assay (RIPA) buffer (1% NP-40, 0.5% sodium deoxycholate and 0.1% SDS in PBS) containing protease, phosphatase and deubiquitinase inhibitors (1 ng/μL aprotinin/leupeptin, 10 mM β-glycerophosphate, 1 mM N-ethylmaleimide, 1 mM Pefabloc). Upon sonication for 15 min, proteins were denatured in 6× Laemmli buffer at 95 °C for 5 min, separated by SDS-PAGE and blotted onto nitrocellulose membranes. The following antibodies were used: anti-H2B (ab1790, Abcam, Cambridge, UK, 1:5000), anti-H2Bub1 (5546, Cell Signaling, Danvers, MA, USA, 1:1000), anti-USP22 (sc-390585, Santa Cruz, Dallas, TX, USA, 1:1000).

The treated osteosarcoma cells were harvested and lysed in a lysis buffer containing protease inhibitor cocktail. The protein concentration was measured by the bicinchoninic acid (BCA) assay. The protein was determined by SDS-PAGE and probed with antibodies against USP22 (SC-390585, Santa Cruz, USA, 1:1000) and tubulin (#T9028, Sigma-Aldrich, St. Louis, MO, USA, 1:5000) as described previously (29 ). The quantitative results were analyzed by ImageJ software (NIH, USA).