Imagequant las3000

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.

Whole-cell extracts or immunoprecipitates were treated with 1 × Laemmli sample buffer (Tris-HCl 125 mm, pH 7.5, 1% sodium dodecyl sulfate, 20% glycerol) and fractionated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The proteins were then transferred to nitrocellulose membranes using an iBlot Transfer Stack and iBlot Gel Transfer Device (Thermo Fisher Scientific, Waltham, MA, USA). After incubation with StartingBlock T20 (phosphate-buffered saline) blocking buffer (Pierce Biotechnology, Waltham, MA, USA), membranes were labeled with primary antibodies overnight, followed by incubation with horseradish peroxidase-conjugated secondary antibodies (Cell Signaling Technology, Danvers, MA, USA). Membranes were incubated with ImmunoStar Zeta (Wako, Osaka, Japan) and signals were detected using ImageQuant LAS-3000 (Fujifilm, Tokyo, Japan).
The following antibodies were used for western blotting analysis: CDT1 (sc-365305; Santa Cruz Biotechnology, Dallas, TX, USA), cleaved PARP (9541; Cell Signaling Technology), γH2AX (2577; Cell Signaling Technology), GAPDH (2118; Cell Signaling Technology), GATA1 (3535; Cell Signaling Technology), PU.1 (2258; Cell Signaling Technology) and c-Jun (9165; Cell Signaling Technology).

Immunoblot analysis for CYGB protein detection was performed essentially as described previously [36] (link) using rabbit anti-CYGB antibody, a kind gift from Dr. Norifumi Kawada [8] (link), and β-actin antibody (Cell Signaling Technology, Beverly, MA). The images were obtained using ImageQuant LAS 3000 (Fujifilm, Tokyo, Japan).