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Characterization of ubiquitin-replaced U2OS cells. Stably transfected U2OS cells (shUb+WT, shUb+K48R, and shUb+K63R) were cultured in the absence (−) or presence (+) of 1 μg/ml tetracycline (Tet) for 72 h before lysis in RIPA buffer. Proteins in the lysates were immunoprecipitated with HA antibody and immunoblotted with antibodies specifically recognizing (A) K48-linked ubiquitin and (B) K63-linked ubiquitin. IP, immunoprecipitation; WB, Western blotting.
