Figure 3

Effect of cell cycle progression on cyclin D1 localization. A. MCF-7 cells were cultured for 48 h in double stripped phenol red-free medium containing 100 nm ICI182,780. Cells were treated with fresh medium containing β17-oestrodiol (100 nm) and harvested at the indicated time points. Cell lysates were separated by 4–20 % SDS-PAGE and immunoblot analysis was done using antibodies against Sp1, GSK3, cyclin D1 and Hsp60. B. MCF-7 cells were left untreated or treated with 1 mM hydroxyurea (HU) for 24 h. HU treated cells were treated with TSA (1 μM) alone or with MG132 (50 μM) for 6 h and treated as in figure 1A. Immunoblot analysis was done using antibodies against Sp1, GSK3, Skp2, cyclin D1 and Hsp60. C. MDA-MB231 cells were treated as in B. D. SKUT-1B cells were treated for 24 h with HU (1 mM) and cell lysates separated by 4–20 % SDS-PAGE. Immunoblot analysis was done using antibodies against Sp1, Skp2, cyclin D1 and Hsp60.