Pk10003

mRNA was transfected into HEK293 cells using a transfection reagent (jetMESSENGER™; PolyPlus, Brant, France). After 24 h, total cell protein and whole-cell supernatants were collected. Each lysate sample underwent sodium dodecyl sulfate–polyacrylamide gel electrophoresis on 10% gels using TRIS-HCl. Then, proteins were transferred onto polyvinylidene fluoride (PVDF) membranes, which were enclosed in TBST (Tris-buffered saline containing Tween 20) solution with 5% skimmed milk. PVDF membranes were treated with primary (rabbit anti-VP7) antibody, followed by addition of a secondary antibody (horseradish peroxidase-coupled goat anti-rabbit IgG; Abcam, Cambridge, UK), followed by incubation for 1 h at room temperature. After washing, treated PVDF membranes were exposed to a highly sensitive luminescence solution (PK10003; Proteintech, Chicago, IL, USA) and imaged using an electrochemiluminescence system (Thermo Fisher Scientific, Waltham, MA, USA).

The liver was grinded for 30 min in the RIPA (Solarbio Life Sciences, Beijing). Then centrifuge the fissile at 12,000 rpm for 10 min. After adding the supernatant to the SDS-PAGE Sample Loading Buffer, the protein was denatured at 98°C for 15 min in a metal bath. The protein sample (60–70 μg) was separated using 7.5% SDS-PAGE and transferred to a PVDF membrane (Millipore, Billerica, MA, USA). The PVDF membrane was subjected to 5% skim milk powder at room temperature for 90 min. The primary and secondary antibodies were then incubated in succession. Antibodies were bought from Abmart (Abmart, Shanghai) and used as recommended by the manufacturer. The primary antibodies CPT1A (TD12004, dilution 1: 1,500), FASN (T56597, dilution 1: 1,500), ACC (T55575, dilution 1: 1,500), PPAR alpha (TA5301, dilution 1: 1,500), β-actin (P30002, dilution 1: 3,000), and Goat Anti-Rabbit Mouse IgG-HRP (M21003, dilution 1: 6,000) were used for target protein determination. Following the manufacturer's instructions, protein bands were seen using an enhanced chemiluminescence kit (Proteintech, PK10003).

Cardiomyocytes were divided into four groups, lysates of cardiomyocytes were prepared in RIPA buffer, and protein concentration was determined by BCA protein assay (Thermo Fisher Scientific, US). Equal amounts (30 μg) of proteins were separated on 10% SDS-PAGE (100 V for 120 min) and transferred into nitrocellulose membranes (120 min at 100 V) using standard procedures. The binding of nonspecific proteins to the membrane was blocked with blocking buffer (5% nonfat milk) at room temperature for 2 h. The membranes were probed with anti-CaMKII (ab52476, Abcam), anti-RIPK1 (ab106393, Abcam), anti-RIPK3 (sc-374639, Santa Cruz), and anti-β-actin (T40104, abmart) at 4°C overnight. After the membrane was washed three times with TBST (20 mM Tris, 500 mM NaCl, and 0.1% Tween 20) for 10 min each time at room temperature, the membrane was incubated with the second antibody (1 : 20000) for 1 h at 37°C. Then, the membrane was washed three times with TBST for 10 min each time at room temperature. Detection was performed using an ECL chemiluminescence kit (PK10003, Proteintech). Finally, the PVDF membrane was placed in the Bio-Rad ChemiDocXRS gel imaging system for photography under exposure, and band intensities were quantified using Image Lab software. Three independent replicates were conducted for this experiment.