Figure 2
From: Dual effects of TGF-β on ERα-mediated estrogenic transcriptional activity in breast cancer
The EREaccommodates Smad3/4 and ERα complexes in the nucleus. (a) MCF-7 cells were treated with E2, E2 + TGF-β1 or vehicle (as a negative control) for 1 hr as indicated. Lysates were subjected to immunoprecipitation with anti-Smad4, anti-Smad2/Smad3 or anti-ERα antibody. The Smad-associated endogenous ERα was detected by immunoblot analysis. The expression levels of endogenous proteins were monitored by immunoblotting of total cell protein. (b) COS-1 cells were transfected with HA-ERα, FLAG-Smad3 and FLAG-Smad4 expression plasmids. Cells were treated with (+) or without (-) E2 and TGF-β1 and subjected to sequential immunoprecipitation, DNA pull-down (DNAP), and immunoblotting. Wild-type (WT) and mutant (mt) biotinylated ERE oligonucleotides were used in the DNAP. The presence of HA-ERα, FLAG-Smad3 and FLAG-Smad4 in the DNAP was detected with anti-FLAG and anti-HA antibodies.