Anti bmp6 antibody

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.

Total protein was extracted, separated by 10% SDS polyacrylamide gel electrophoresis and transferred to PVDF membrane (Millipore, Bedford, MA, USA). Membranes were blocked with 5% non-fat dry milk in TBST and were incubated with following primary antibodies: anti-MNAT1 antibody, anti-EIF2A antibody, anti-KSR1 antibody, anti-ADCY9 antibody and anti- kLRP1 antibody (1:1000 Abcam, Cambridge, UK), anti-PAM16 antibody, anti-SF3A3 antibody, anti-LSM5 antibody and anti-TRAM2 antibody (1:800 Abcam, Cambridge, UK), anti-BMP6 antibody (1:400 Abcam, Cambridge, UK), anti-GAPDH antibody (1:2000 Bioworld, Minnesota, USA) at 4 °C overnight. Secondary antibodies were added, and the bands were visualized using the enhanced chemiluminesczence (Amersham Biosciences, Buckinghamshire, UK). GAPDH was used as the loading control.