For immunoblotting analysis, cells were harvested by scraping, washed in PBS, resuspended, then homogenized and sonicated in RIPA buffer (1× PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM sodium orthovanadate, 10 mg/mL aprotinin, and 100 mg/mL phenylmethyl sulfonyl fluoride [PMSF]). Lysates were then centrifuged at 12,000g for 10 minutes at 4°C, and supernatants were collected for analysis. The protein concentrations were measured using a commercial protein assay (Bio-Rad Laboratories, Hercules, CA) before Western blot analysis. Protein from 661W cells and MCF7 cells was mixed with loading buffer, boiled for 5 minutes, separated by SDS-PAGE, and then transferred to polyvinylidene difluoride (PVDF) membranes (Immunobilon-P; Millipore, Bedford, MA). Membranes were blocked with 5% dry milk in PBS. Proteins were probed with specific ERα or ERβ antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) at 1:1000 dilution and incubated overnight at 4°C. The blots were washed and applied with goat-anti-rabbit secondary antibodies (Santa Cruz Biotechnology) at 1:1000 dilution. After washing, the blots were developed with an enhanced chemiluminescent kit (Pierce, Rockford, IL).