Figure 6.

Expression of ERβ in MDA-MB-231 cells stably transfected with PGRMC1 (PG). A and B, Detection of ERβ mRNA (A) in PG- and vector (V)-transfected cells by RT-PCR and ERβ protein in transfected cell membranes (B) by Western blot analysis. C and D. Immunocytochemistry of ERβ protein in V (C) and PG cells (D). E, Western blot analysis of biotinylated cell-surface proteins with the ERβ antibody. F, Detection of ERβ in different subcellular fractions of PG and V cells (upper) and specific [³H]E2 binding to PG cell membranes in a single point assay (lower). G, Representative saturation curve and Scatchard plot of specific [³H]E2 binding to plasma membranes of PG cells. H, Effects of transfection of PG cells with ERβ siRNA (si-ERβ) or nontarget siRNA on membrane ERβ mRNA and protein expression (top) and specific [³H]E2 binding in a single point binding assay (bottom). M, molecular weight marker; Mm, plasma membrane fraction; Ms, microsomal; fraction; Cy, cytoplasmic fraction; N, nuclear fraction; V1,V2, PG1, PG2, separate clones of transfected MDA-MB-231 cells. **, P < .01; *, P < .05 compared with V or nontarget controls; n = 6. All the experiments were repeated 3 or more times and similar results were obtained in each experiment