Fusion molecular imaging system

Western blotting was performed as described previously [46 (link)]. Aliquots of 15 μg cellular lysate protein were resolved by SDS-polyacrylamide gel electrophoresis (10% resolving gel) and subsequently transferred to a nitrocellulose membrane (AmerSham Biosciences, USA). Membranes were incubated with phospho-ERα (Ser122; Biorbyt, Cambridge, UK), phospho-ERα (Ser167; Abcam, UK), ERα (Abcam), phospho-GSK-3β (Ser9; Cell Signaling, Danvers, MA, USA), GSK-3β (Cell Signaling), active-β-catenin (Millipore, Molsheim, France), β-catenin (Millipore), and RUNX2 (Cell Signaling) antibodies overnight at 4 °C, respectively. To demonstrate equivalency of protein loading, a specific GAPDH antibody (Cell Signaling) was used. The membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit, rabbit anti-sheep or goat anti-mouse secondary antibody (Cell Signaling) for 1 h at room temperature. For the enhanced chemiluminescence reaction, immunoblots were developed in SuperSignal West Pico Chemilumemescent Substrate (Perbio Science, Pierce, Bonn, Germany), and the respective phosphoproteins or total proteins were visualized using the Fusion Molecular Imaging System (Vilber Lourmat, Eberhardzell, Germany).

We resolved 10 µg of cellular lysate protein using SDS-PAGE and transferred it to a nitrocellulose membrane (BioRad, Hercules, USA). The membranes were incubated with anti-GAPDH, Mdk (both Santa Cruz Biotechnology, Dallas, USA), β-catenin (EMD Millipore Corporation, Merck, Darmstadt, Germany) or collagen type 2 (Rockland, Pennsylvania, USA) antibodies overnight at 4°C, respectively. The membranes were incubated using horseradish peroxidase-conjugated secondary antibodies and developed in SuperSignal West Pico Chemiluminescent Substrate (Perbio Science, ThermoFisher Scientific, Waltham, USA). The protein bands were visualized using the Fusion Molecular Imaging System (Vilber Lourmat, Eberhardzell, Germany).