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Fig. 6. p130Cas modulates estrogen-dependent cyclin D1 expression. (A) T47D cells were transiently transfected with empty vector or the myc-tagged p130Cas cDNA by the Lipofectamine 2000 method. 24 hours post transfection, cells made quiescent (as indicated in Fig. 1) for an additional 24 hours, were left untreated or treated with 10 nM E2 for the indicated times and detergent extracted. Cell extracts were run on 10% SDS-PAGE, transferred to nitrocellulose, blotted with cyclin D1 antibodies (upper panels) and re-blotted with polyclonal antibodies to myc epitope (lower right panel). (B) T47D cells were treated with 10 nM E2 for the indicated times in the presence of either 5 µM PP1 or 25 µM PD98059 and detergent extracted. Cell extracts were run on 10% SDS-PAGE, transferred to nitrocellulose and blotted with anti-cyclin D1 antibodies. (C) T47D were depleted of p130Cas by siRNA transfection as indicated in Fig. 5. After 48 hours, cells were treated with 10 nM estrogen for the indicated times and detergent extracted. Cell extracts were run on 10% SDS-PAGE, transferred to nitrocellulose, blotted with p130Cas antibodies (upper panel) or with cyclin D1 (lower panel). Similar results were obtained in three independent experiments.
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