Figure 2.

Expression of estrogen receptors (ERs) in human endothelial cells. (A) mRNA levels of ER-α and ER-β (Real time PCR). Relative mRNA levels of ER-α and ER-β were calculated as a value of the cycle threshold (C_t), which was normalized to GAPDH mRNA levels (C_t values) by calculating the 2^{[Ct(GAPDH) − Ct(ER)]} value, simply termed 2^−∆Ct. For each cell type, mRNA level of ER-β was standardized as 100% (internal control), while the mRNA level of ER-α was compared with that of ER-β; (B) Protein levels of ER-α and ER-β (Western blot). A protein (30 μg) sample from each cell line was loaded in the each well of SDS-PAGE (10% polyacrylamide) for electrophoresis. ER-β (59 kDa) and ER-α (65 kDa) protein bands were detected by their specific antibodies. The same blot with the same protein loading was used for detecting both ER-β and ER-α proteins. The density of Western blot bands was analyzed by using NIH ImageJ software. Thus, a positive ER-β band could serve as an internal control for ER-α, which had no band. For each cell type, protein level of ER-β was standardized as 100% (internal control), while the protein level of ER-α was compared with that of ER-β. HCAEC (human coronary artery endothelial cells); HUVEC (human umbilical vein endothelial cells); HPAEC (human pulmonary artery endothelial cells); EA.hy 926 (Immortalized HUVEC cell line); PCR (polymerase chain reaction). WB (Western blot). Human endothelial cells express mainly ER-β and no or very small amounts of ER-α.