Iron chelators decrease general (Ub)_n and cyclin D1 polyubiquination when proteasome activity is inhibited. (A) General protein polyubiquitination is decreased when cells are incubated with 311 in the presence of the proteasomal inhibitors MG or LC. MCF-7 cells were incubated with media alone or medium containing 311 (25 μM) in the presence and absence of MG (20 μM) or LC (10 and 20 μM) for 24 hours at 37°C. Total protein was extracted from cells and Western analysis was performed. (B) 311 decreases cyclin D1 protein expression and also decreases polyubiquitination of cyclin D1 in the presence of MG. MCF-7 cells were incubated with either media alone or media containing 311 (25 μM) in the presence and absence of MG (20 μM) for 8, 18, and 24 hours. Immunoprecipitation was performed using anti–cyclin D1 antibody. Complexes were then separated by SDS-PAGE and probed with anti–cyclin D1 or antiubiquitin antibody using Western analysis. Results shown are typical experiments from 3 performed. The horizontal broken line in the densitometric analysis represents a value of 100%.