Chemiscope 3000

Cardiomyocytes in each group were collected and total proteins were extracted using RIPA RIPA Lysis and Extraction Buffer (AR0105, Boster Biological Technology, Wuhan, China), and the protein concentration was determined using Easy II Protein Quantitative BCA kit (DQ111-01, TransGen Biotech, Beijing, China). The OD values were measured at 562 nm, and the concentration of proteins were calculated with the standard curve. The protein samples were pre-processed using 5×SDS-PAGE loading buffer (with β-Mercaptoethanol) (C05-03001, Bioss, Beijing, China), isolated with electrophoresis (10% gel for eIF2α and GRP78; 12% gel for ATF4 and β-actin; 15% gel for CHOP, BCL-2 and BAX), and transferred to ploy membrane (0.45 um polyvinylidene fluoride PVDF membrane [IPVH00010, Millipore, the USA] for eIF2α, ATF4, GRP78 and β-actin; 0.22 um PVDF membrane [ISEQ00010, Millipore, the USA] for CHOP, BCL-2 and BAX). The membrane was blocked using blocking solution containing 5% skim milk for 1 h, and incubated with primary and secondary antibodies (Table S1). Mouse β-actin was used as the internal reference for other proteins. The protein bands were visualized using chemiluminescence kit (Chemiscope 3000, Clinx Science Instruments, Shanghai, China) to assess the expression levels of eIF2α, ATF4, CHOP, GRP78, BCL-2 and BAX in each group.

After treatment shown above was over, cells were washed with PBS three times and were lysed with RIPA lysis buffer supplemented with the protease inhibitor (PMSF) and incubated on ice for 30 minutes. Supernatant was collected after centrifugation (12,000 rpm, 15 min), and protein concentration was determined by using the BCA protein assay kit according to the manufacturer's recommendations. The lysate was boiled with loading buffer and separated with 10% sodium dodecyl sulfate polyacrylamide gels (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes. After blocking with blocking buffer (5% skim milk powder in TBST (0.1% Tween 20 in Tris-buffered saline (TBST)) for 1 h, primary antibody 6 × HisTag (mouse polyclonal, Cell Signaling Technology, Boston, MA; 1:1000) was incubated overnight at 4 °C. After washing PVDF membrane three times with TBST, goat anti-mouse secondary antibody (Santa Cruz Biotechnology, Dallas, TX; 1:1000) coupled with horseradish peroxidase (HRP) was incubated for another 1 h at room temperature. Anti-β-actin-HRP (Santa Cruz Biotechnology, Dallas, TX; 1:1000) was used as a loading control. Chemical reaction light signal detection was performed using the Clinx ChemiScope 3000 mini enhanced chemiluminescence (ECL) detection reagent.