Bicinchoninic acid reagent kit

The whole protein of rat left ventricular tissues or cardiomyocytes was extracted using the RIPA lysis with PMSF. A bicinchoninic acid reagent kit (Beyotime, Shanghai, China) was used to determine the protein concentration. The proteins were separated in 10% gels using a sodium dodecyl sulphate-polyacrylamide gel electrophoresis and then transferred onto polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA). The membranes were blocked with 5% non-fat milk at room temperature for 2 h and then incubated at 4°C overnight with the specific primary antibodies: anti-GRP78 (1:1000), anti-p-PERK (1:1000), anti-PERK (1:1000), anti-CHOP (1:1000), anti-cleaved caspase 3 (1:1000), anti-caspase 3 (1:1000), anti-IRE1 (1:1000), anti-p-IRE1 (1:1000), anti-ATF6 (1:1000), anti-α2A-AR (1:200), anti-α2B-AR (1:2000), and anti-GAPDH (1:3000). Next, the membranes were washed and incubated at room temperature for 2 h with the HRP-conjugated secondary antibodies (1:5000, Santa Cruz, CA, USA). For each sample, three replicates were tested. The bands were detected using an enhanced chemiluminescence kit (NCM, Suzhou, China) under a Tanon 5200 luminescent imaging workstation (Tanon, Shanghai, China). The protein intensity was analyzed with the Image J software and normalized to GAPDH as control.

Total proteins from MC3T3-E1 cells or bone tissues of mice were extracted with RIPA lysis buffer containing protease inhibitors (Sigma–Aldrich). The protein concentration was quantified by the bicinchoninic acid reagent kit (Beyotime, China). The proteins were separated by 10% SDS-PAGE and transferred to Immuno-Blot PVDF membranes (Merck Millipore, Germany). The membranes were then blocked with 5% BSA and overnight incubated with specific primary antibodies targeting BSEP (1:1000, Abcam, ab155421), RUNX2 (1:1000, Cell signalling Technology, 12,556), and GAPDH (1:3000, Abcam, ab8245) at 4 ​°C. The next day, the blots were then incubated in secondary antibody (horseradish peroxidase–labelled) for 1 ​h with shaking at room temperature, and the blots were visualized by SuperSignal chemiluminescent HRP substrates (Thermo Scientific) using Molecular Imager ChemiDOC™ XBS imaging systems (Bio-Rad Laboratories).