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Fig. 4. p130Cas over-expression modulates estrogen-dependent Erk1/2 MAPK activation. (A) T47D cells were grown to confluence and made quiescent (as indicated in Fig. 1) for 24 hours, left untreated or treated with 10 nM E2 for the indicated times and detergent extracted. Erk1/2 MAPK activation was measured and the relative amount of MAPK activation was calculated by densitometric analysis (on the right) as explained in Fig. 3. (B) T47D cells in the same conditions as in A were treated with 10 nM estrogen in the presence of 5 µM PP1 for the indicated times. (C) Alternatively, T47D cells were transiently transfected either with empty vector or the p130Cas cDNA using Lipofectamine 2000. 24 hours post transfection, cells were made quiescent in phenol red-free RPMI-1640 medium supplemented with charcoal-treated serum for an additional 24 hours, left untreated or treated with 10 nM E2 for the indicated times and detergent extracted. Densitometric analysis (right) was performed as previously described and the statistical significance was calculated using Student's t-test (*) P<0.05. (A-C) Cell extracts were run on 10% SDS-PAGE, transferred to nitrocellulose, blotted with anti-phospho Erk1/2 antibodies (upper panels) and re-blotted with polyclonal antibodies to Erk1/2 (lower panels in A and B) and c-myc (lower panel in C). Similar results were obtained in four independent experiments.
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