Fig. 3. Irofulven treatment is accompanied by polyubiquitylation of Pol II LS followed by its proteasome-mediated proteolysis. (A) HeLa cells were incubated in the absence or presence of 1 µg/ml irofulven for 30 minutes, polyubiquitylated proteins were affinity purified and the resulting proteins were subjected to gel electrophoresis and the blots probed with the H14 monoclonal antibody. (B) HeLa cells were incubated in the absence or presence of 1 µg/ml irofulven for 30 minutes, Pol II LS was immunoprecipitated and the resulting proteins were subjected to gel electrophoresis and the blots probed with the P4D1 monoclonal antibody against ubiquitin. (C) HeLa cells were treated for the indicated times with 1 µg/ml irofulven in the absence (upper panel) or presence (lower panel) of 5 µM MG-132. (D) HeLa cells were treated for 6 hours with 1 µg/ml irofulven in the absence or presence of increasing amounts of velcade (bortezomib/PS-341) ranging from 20 to 1000 nM. (E) HeLa cells were incubated with 1 µg/ml irofulven for the indicated times in the presence () or absence () of 100 µM DRB. The total amounts of Pol II LS were assessed by immunoblotting and the relative intensities measured and standardized with respect to actin levels. Error bars represent standard errors and are indicated when they exceed symbol size.