Figure 3.

ERβ does not translocate to nucleus. A, NSCLC cells and MCF-7 cells were transiently transfected with ERE-TK-luc and pRLTK-luc plasmids. After 24 h, cells were treated with or without E2 overnight and subjected to luciferase assays. Results are expressed as relative luciferase units, the ratio of firefly to Renilla luciferase. Values are shown as means (±sd) from three identical wells. B, Nuclear and cytosolic fractions were prepared from NSCLC cell lysates and subjected to Western blotting with ERβ antibodies (Upstate Biotechnology). PARP was used as loading control for nuclear fractions. C, NSCLC cells grown in phenol red free media were treated with E2 (100 nm) for 1 h. Nuclear and cytosolic fractions were prepared, and ERβ was analyzed by Western blotting with antibodies from Upstate Biotechnology. PARP and β-actin were used as loading controls for nuclear and cytosolic fractions, respectively. D, Western blotting analysis in H1299 cells stably expressing ERα. E, H1299 cells stably expressing empty vector (EV) or ERα constructs were transfected with ERE-TK-luc and pRLTK-luc plasmids, and luciferase assays were performed as described above. Values are shown as means (±sd) from three identical wells. C, Cytoplasmic; N, nuclear.