Figure 5

Western blotting of whole-cell lysates of treated MCF-7 and T47D cells for the expression of Cyclin D1. (A) MCF-7 cells were treated with ATRA or RAMBAs for 6 days or 4HPR for 4 days and then cell lysates were electrophoresed using 15% SDS–PAGE and subjected to Western blotting. Lane 1: control; lanes 2–4: VN/14-1 (1, 5 and 10 μ M); lane 5: ATRA (5 μ M); lane 6: VN/50-1 (5 μ M); lane 7: VN/66-1 (5 μ M); lane 8: VN/69-1(5 μ M); and lane 9: 4-HPR (5 μ M). (B) T47D cells were treated with VN/50-1 and VN/14-1 for 6 days and then cell lysates were electrophoresed using 15% SDS–PAGE and subjected to Western blotting. Lane 1: control; lanes 2–4: VN/50-1 (1, 5 and 10 μ M); and lanes 5–7: VN/14-1 (1, 5 and 10 μ M). Numbers below the blot show fold decrease in expression of the protein as analysed by ImageQuant densitometry analysis. Membranes were stripped and probed for β-actin to verify equal protein loading. Primary antibody (Cell Signaling Technology) 1 : 2000 in 10% milk TBST overnight at 4°C and secondary antibody (anti-rabbit) 1 : 2000 in 10% milk TBST for 1 h at RT. The experiments were repeated twice with similar results.