Tubulin

Frozen ventricular tissue samples were homogenized in RIPA buffer (Beyotime Biotechnology, China) containing protein enzyme inhibitors, and protein concentration was quantified using a BCA Protein Assay Kit (Beyotime Biotechnology, China). The lysates were separated on 8–15% SDS-PAGE gels and then electro-transferred to 0.45μm polyvinylidene difluoride membranes (Millipore, USA). After blocking with 5% nonfat milk in Tris-buffered saline at room temperature for 2h, the membranes were incubated for overnight at 4°C with primary antibodies. The primary antibody included IL-6 (Wanlei, China), IL-1β (Affinity, China), IL-18 (Affinity, China) and Tubulin (Affinity, China). After washing three times for 10min per wash, the membranes were incubated with an HRP-conjugated secondary antibody for 2h at room temperature. Subsequently, the blots were imaged using a Bio-Rad imaging system (Bio-Rad, USA) and protein expression levels were determined using ImageJ software.

Proteins were resolved by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to polyvinylidene fluoride (PVDF) membranes, and blocked in 5% skimmed milk for 1 h. The membranes were then incubated overnight at 4°C with the following primary antibodies (anti-rabbit or anti-mouse): cleaved caspase3 (1 : 1,000; Affinity, China), Tom20 (1 : 1,000; BD, USA), COXIV (1 : 1,000; Affinity, China), LC3B (1 : 500; Affinity, China), P62 (1 : 1,000; Affinity, China), Bcl-2 (1 : 1,000; Affinity, China), Bad (1 : 1,000; Affinity, China), Parkin (1 : 1,000; Proteintech, USA), PINK1 (1 : 1,000; Proteintech, USA), Drp1 (1 : 1,000; Affinity, China), Mfn2 (1 : 1,000; Affinity, China), Atg-5 (1 : 800; Affinity, China), β-actin (1 : 5,000; Affinity, China), GAPDH (1 : 5,000; Affinity, China), Bcl-xl (1 : 1,000; Affinity, China), Bcl-w (1 : 1,000; Affinity, China), Bim (1 : 1,000; Affinity, China), procaspase3 (1 : 1000; Abcam, USA), and Tubulin (1 : 5,000; Affinity, China). Membranes were then incubated for 2 h with horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG secondary detection antibodies (1 : 5,000). Immunoreactivity was detected by using an enhanced chemiluminescence detection system (Beyotime, Haimen, China) and visualized on an imaging system (Kodak, Shanghai, China).

Liver tissues from wild-type (wt) and NASH model mice were finely minced and then subjected to protein extraction via the RIPA method. The expression levels of β-actin and tubulin were normalized using their grayscale values measured by ImageJ. Polyacrylamide gels were prepared using the One-Step PAGE Gel Fast Preparation Kit (15%) from Vazyme (catalog number E305), with the 180 kDa Prestained Protein Marker from Vazyme (catalog number MP102) used for molecular weight estimation. Electrophoresis and membrane transfer were conducted using the PowerPac Basic Power Supply from BIO-RAD. Blocking was performed with 5% BSA. Primary antibodies were diluted as follows: β-actin at 1:1000 from Servicebio (catalog number GB15001-100), tubulin at 1:5000 from Affinity Biosciences (catalog number T0023), CDKN1B at 1:1000 from BIOSS (catalog number bs-0742R), and TFAM at 1:1000 from Proteintech (catalog number 22586-1-AP). Imaging was done using the Tanon 4800 system. Grayscale values for all bands were acquired with ImageJ, and the relative protein expression levels were determined using β-actin and tubulin as standards. Statistical analysis and graphical representation were performed using GraphPad Prism 9.