Glyceraldehyde 3 phosphate dehydrogenase gapdh

After treatments, proteins were extracted from H9c2 cells or heart tissues lysed in a RIPA buffer containing 1% PMSF and 1% phosphatase inhibitor cocktail (Solarbio). The protein concentration was determined using a BCA protein assay kit (Solarbio). Equal protein extracts were analyzed by Western blotting, with primary antibodies against Bax, GRP78, GRP94, and PDIA6 (Abcam, Cambridge, U.K.); Bcl-2 (R&D Systems, Minneapolis, MN, U.S.A.); Caspase-12, ATF6, PERK, and p-PERK (Proteintech Group, Chicago, IL, U.S.A.); CHOP, cleaved Caspase-3, AMP-activated protein kinase (AMPK), and p-AMPK (Cell Signaling, Danvers, MA, U.S.A.); IRE1α, p-IRE1α, and peroxisome proliferator-activated receptor γ coactivator 1α (PGC1α) (Novus Biologicals, Littleton, CO, U.S.A.); and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; ImmunoWay, Plano, TX, U.S.A.). Immunoblots were detected by ECL (Millipore, Billerica, MA, U.S.A.) with anti-mouse or anti-rabbit IgG coupled to horseradish peroxidase as the secondary antibody (ImmunoWay). Gel images were captured using a Universal Hood II (Bio-Rad, Hercules, CA, U.S.A.) and quantitated with the ImageJ 1.51k software.

The supernatant (4 μg/μL) mixed with a certain proportion of 5 ×  and 1 ×  SDS-PAGE loading buffer (Solarbio, China) was boiled at 95°C for 10 min. Equal amounts of protein (50 μg/20 μL) were separated by 10% SDS-PAGE (SDS-PAGE Gel Kit, Solarbio Life Sciences, China). Proteins were electrophoretically transferred to polyvinylidene difluoride membranes (Bio-Rad, Richmond, USA), which were then blotted with an antibody against ATP synthase subunit epsilon (ATP5E) (ThermoFisher, USA) diluted 1 : 500, voltage-dependent calcium channel subunit alpha-2/delta-1 (CACNA2D1) (Abcam, Hong Kong) diluted 1 : 10000, glutathione peroxidase 3 (Gpx-3) (Santa, USA) diluted 1 : 2000, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Immunoway, USA) diluted 1 : 20000. A secondary antibody (Abcam, Hong Kong) was used to detect the primary antibody. Anti-beta actin antibody (Immunoway, USA) and secondary antibody (Abcam, Hong Kong) were used as references. Finally, chemiluminescence analysis, development, and fixation were carried out. Band signals were detected using an iBright CL750 western blot imaging system (ThermoFisher, USA) and analyzed using iBright Software (ThermoFisher, USA).