Fig. 4.

K48- and K63-specific ubiquitin linkages are dispensable for IDOL autoubiquitination and autodegradation. A: U2OS cell lines were cultured in the absence (−) or presence (+) of 1 μg/ml tetracycline (Tet) for 48 h before infection with adenovirus encoding IDOL. Cells were lysed in RIPA buffer for 24 h after the adenovirus infection. Proteins in the lysates were immunoblotted with an IDOL monoclonal antibody. B: U2OS cells were cultured in the presence of 1 μg/ml tetracycline for 48 h before infection with adenovirus encoding IDOL. Cells were lysed in RIPA buffer for 24 h after the adenovirus infection, with the last 4 h in the presence of 25 μM MG132. Proteins in the lysates were immunoprecipitated with an antibody-recognizing IDOL and immunoblotted with HA antibody. C: U2OS cells were cultured in the absence or presence of 1 μg/ml tetracycline for 48 h before being infected with adenovirus encoding IDOL. Cells were lysed in RIPA buffer for 24 h after adenovirus infection, with the last 4 h in the presence of 25 μM MG132. Proteins in the lysates were immunoblotted for IDOL. IP, immunoprecipitation; WB, Western blotting.