Anti bmper antibody

Mouse livers were fixed in 10 % neutral-buffered formalin for 24 h at 4 °C and processed for paraffin embedding. The paraffin sections were prepared and subsequently dehydrated in graded ethanol and xylene. The sections were stained using hematoxylin and eosin (H&E), Masson’s trichrome or Berlin blue staining protocols and were immunostained with an anti-BMPER antibody (Abcam plc, Cambridge, UK).

Proteins were extracted from the mouse livers using RIPA buffer (150 mM NaCl, 0.25 % deoxycholic acid, 0.1 % sodium dodecyl sulfate [SDS], 50 mM Tris–HCl, pH 8.0). The protein concentration of each sample was determined by the Bradford method using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA), according to manufacturer protocols. A total of 30 μg protein was subjected to electrophoresis using 12 % Mini PROTEAN TGX™ precast gels (Bio-Rad Laboratories, Hercules, CA, USA). The proteins were then transferred to nitrocellulose membranes (Bio-Rad), blocked in SuperBlock blocking buffer (Thermo Fisher Scientific) for 1 h at room temperature, and incubated with anti-SMAD1 rabbit antibody (Cell Signaling Technology [CST], Danvers, MA, USA), anti-phospho-SMAD1/5/8 rabbit antibody (CST), anti-β-actin mouse antibody (BD Biosciences), anti-BMPER antibody (Abcam) or anti-BMP6 antibody (Abcam) overnight at 4 °C. The membranes were incubated with horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG (R&D Systems, Minneapolis, MN, USA) and visualized using the SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific). The membranes were photographed with ImageQuant LAS 3000 (Fujifilm, Tokyo, Japan), and the band densities were analyzed using ImageJ software.