Ecl western blotting substrate

Total RNA from frozen liver samples or HepG2 cells was isolated using TRIzol reagent and real-time quantitative PCR was performed as previously described [11] (link), [41] (link). Primer sequences for qPCR are shown in Table S1. Total protein was isolated from frozen liver samples and HepG2 cells and Western blotting was performed as previously described [11] (link), [41] (link). Protein detection was performed using polyclonal rabbit antibodies to total and phosphorylated cFos, cJun, ERK, and JNK (Cell Signaling Technology, Danvers, MA). Bound antibody was detected using goat anti-rabbit polyclonal HRP antibody (Cell Signaling Technology) and developed using ECL Western Blotting Substrate (Cell Signaling Technology). Representative Western blots of pooled samples are shown.

Total RNA from murine liver and harvested HepG2 cells was isolated using TRIzol reagent and real‐time quantitative PCR was performed as described previously.²⁴, ²⁵ Total protein was isolated and western blotting was performed as described previously.²⁴, ²⁵ Protein detection was performed using polyclonal rabbit antibodies to total and phosphorylated JNK and ERK (Cell Signaling Technology). Bound antibody was detected using goat anti‐rabbit polyclonal HRP antibody (Cell Signaling Technology) and developed using ECL Western Blotting Substrate (Cell Signaling Technology). Representative Western blots of pooled samples are shown.

Cells were lysed in RIPA buffer (Beyotime, China) combined with a cocktail of protease inhibitors (Thermo Scientific, Rockford, IL). Equal amounts of proteins (25 μg) of different groups were separated by 10% SDS-PAGE and transferred to 0.45-μm PVDF membranes (Millipore, USA). The membrane was first blocked with 5% BSA for 1 h at room temperature and incubated at 4 °C overnight with primary antibodies: GAPDH (1:1000; Abcam, Cambridge, UK), RUNX2(1:1000; Cell Signaling Technology, Danvers, MA), DSPP (1:1000; Abcam, Cambridge, UK), and DMP-1 (1:1000; Abcam, Cambridge, UK). After washed with Tris-buffer saline containing 0.05% Tween 20 (TBST) for three times and 5 min each, the membranes were incubated with secondary antibodies labeled with horseradish peroxidase (1:3000; Cell Signaling Technology, Danvers, MA) at room temperature for 1 h. Blots were visualized using ECL Western Blotting Substrate.