Figure 3.

Identification of membrane estrogen receptor (mER) in caveolae by Western blot analysis. (a) Caveolin-1 (upper panel) and caveolin-2 (lower panel) in MCF-7 cell membranes. Membrane fractions containing caveolae were separated on discontinuous sucrose gradients. Fractions 5 and 6 were combined and 20 and 40 μg/ml protein (shown by adjacent lanes with connecting bar) were resolved on a 4–20% SDS-polyacrylamide gel, transferred to a nitrocellulose membrane, and probed with specific antibodies. Fractions from human endothelial (HE) and mouse fibroblast (RSV-3T3) cells were used as positive controls (50 μg/ml protein). (b) Whole cell lysates (GH₃, MCF-7) and pooled membrane fractions 5 and 6 (Fr.5+6) from sucrose gradients for MCF-7 cells were probed with estrogen receptor-α-specific antibody C-542 (panel 1) or the same antibody blocked with the epitope peptide (panel 2). The arrows show the positions of major bands that were identified as receptor by epitope competition (at 67 and 52 kDa).