*2.6. Western Blot Analysis*

A western blot analysis was performed to examine the expression levels of the proteins. Equal quantities (30 μg) of protein were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto nitrocellulose membranes. After transfer, membranes were blocked with Tris-buffered saline (TBS) containing 0.1% Tween-20 (TBST) and 5% non-fat-milk for 1 h. The membranes were then incubated with specific primary antibodies (Cell Signaling Technology, Danvers, MA, USA): Anti-cyclin D1 (1:1000), anti-p-ERK 1/2 (1:1000), anti-ERK 1/2 (1:1000), anti-ERα (1:1000), anti-SRC-1 (1:1000), and anti-β-actin (1:5000) overnight at 4 ◦C. After washing three times with TBST for 30 min, membranes were incubated with an anti-rabbit (1:80000) or anti-mouse (1:5000) immunoglobulin G (IgG) secondary antibody (Sigma) for 1 h, and then washed with TBST three times for 30 min. Immunoreactive proteins were detected by enhanced chemiluminescence (ECL) (Bionovas, Toronto, Canada) Western blot detection system.
