Figure 6

Regulation of salinomycin-induced Skp2 degradation in DXR cells. (a) Cells were treated without or with 4 μM salinomycin for 24 h, and equal amounts of cell lysates were subjected to immunoblot analysis using the indicated antibodies. (b) DXR cells were treated without or with 4 μM salinomycin for 24 h, and the levels of Cdh1 mRNA were determined by qPCR. The levels of Cdh1 mRNA were normalized to mRNA levels of GAPDH and B2M. (c) DXR cells were treated without or with 4 μM salinomycin for 24 h, and cell lysates were separated into cytosolic and nuclear fractions. Protein samples of cytosolic and nuclear fractions were analyzed by immunoblot analysis using the indicated antibodies. PARP and α-tubulin were used as controls for the nuclear and cytosolic fractions, respectively. (d) DXR cells were transfected with si-Cont or si-Cdh1 for 24 h and treated without or with 4 μM salinomycin for 24 h. Equal amounts of cell lysates were subjected to immunoblot analysis using the indicated antibodies. (e) DXR cells were treated as in panel d, and cell lysates were subjected to immunoprecipitation using anti-Skp2 or normal rabbit IgG antibodies. Immunoprecipitates were processed for immunoblot analysis using ubiquitin and Skp2 antibodies. (f) DXR cells were treated as in panel d, followed by cell cycle analysis. The percentage of cells in G1 phase is annotated in each column. Data are presented as averages of triplicate measurements, with error bars representing standard deviations. These experiments were performed three times and yielded comparable results