Ab133526

MCF-7 cells were cultured in DMEM with 5% charcoal-dextran stripped FBS for 1 day to eliminate any estrogenic source before treatment, then the cells were treated with QYFE (0.00001~0.01mg/mL), 17β-estradiol (0.01 μM) with or without 0.1 μM ICI182, 780, 0.1% DMSO treatment as negative control. After 48h, all cells were harvested protein. Rabbit anti-ER α monoclonal antibody (dilution 1/1000, ab32063, Abcam Biotechnology, UK), rabbit anti-ERβ polyclonal antibody (dilution 1/2000, ab3577, Abcam Biotechnology, UK), rabbit anti-pS2 monoclonal antibody (dilution 1/1000, ab92377, Abcam Biotechnology, UK), rabbit anti-PR monoclonal antibody (dilution 1/1000, ab133526, Abcam Biotechnology, UK) were used, and rabbit anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) monoclonal antibody (dilution 1/5000, ab181602, Abcam Biotechnology, UK) was used as internalcontrol. Finally incubated with goat anti-Rabbit IgG polyclonal antibodyHRP-Labelled (1:4000; C1309; Beijing Pulilai Gene Technology Co., Ltd.). All determinations were carried out in triplicate. Visualization of protein bands was accomplished using Immobilon Western Chemiluminescent HRP Substrate (Millipore, Billerica, MA, USA) [39 (link)].

Western blot was performed as described previously [28 (link)]. The primary antibodies were as follows: anti-IGFBP1 (ab180948, Abcam), anti-Rev-erbα (sc-393215, Santa Cruze), anti-β-Actin (ab179467, Abcam), anti-β-Tubulin (ab179513, Abcam), anti-PR (human, 8757, Cell Signaling Technology), anti-C/EBPβ (ab32358, Abcam); anti-IL-6 (human, ab233706, Abcam), anti-IL-6R (human, ab222101, Abcam), anti-PR (mouse, ab133526, Abcam), anti-IL-6 (mouse, ab229381, Abcam), anti-IL-6R (mouse, ab300581, Abcam), anti-Wnt4 (sc-376279, Santa Cruze). β-Tubulin and β-Actin were used as internal standards.