Fig. 4.

ERβ regulates Cdc25A expression. (a) T47D ERβ cells were spread on six-well plates at a low confluency of 40% and synchronized as described in Materials and Methods. Tetracycline was removed 12 h before start of treatment with 10 nM E2. Cells were harvested in TRIzol at different time points, and cDNA for real-time PCR was prepared. (b) Whole-cell extracts were prepared from synchronized T47D ERβ cells grown on 100-mm plates as described in Materials and Methods. Proteins (50 μg) were separated on SDS/PAGE, electrotransferred to nitrocellulose membrane, and detected with antibody directed against Cdc25A (mouse monoclonal Ab-3, NeoMarkers Fremont, CA). Evaluation of signal strength was done with Bio-Rad chemidoc 1.0 (gel-imaging system).