Fig. 4. Cytoplasmic IN is subject to proteasome-dependent degradation. (A) HeLa cells (Mock) or cells stably expressing IN (IN) were treated with DMSO (–) or 5 µM MG-132 (+) for 5 hours. Following this treatment, nuclear and cytoplasmic extracts were prepared and an equal fraction of each was analysed by western blotting with the 12CA5 antibody. The stabilized IN predominantly localized to the cytosolic fraction, although a slight increase in nuclear staining was also observed. We note that the apparent cytoplasmic abundance of IN by fractionation was due to incomplete extraction of IN from nuclear lysates (data not shown) (Cherepanov et al., 2003). (B) HeLa cells were mock transfected (–) or transiently transfected with IN (IN). 18 hours later, cells were pulsed with 5 µM MG-132 for 5 hours. Subsequently, whole cell extracts were prepared and immunoprecipitated with anti-FLAG or anti-ubiquitin (Ub) antibodies. High-molecular-weight ubiquitin conjugates were detected in IN immunoprecipitates by anti-Ub western blotting (left). Western blotting with the anti-FLAG antibody confirms the expression and immunoprecipitation of IN (right). Following immunoprecipitation of whole cell extracts with anti-Ub antibodies, IN was detected by anti-FLAG western blotting (right). Abbreviations: IgH, immunoglobulin heavy chain; IgL, immunoglobulin light chain. Owing to the high levels of antibody used for the rabbit anti-Ub immunoprecipitation, the FLAG antibody cross-reacted with the rabbit heavy chain.

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