Ecl western blotting substrate

Total protein from MAC-T cells was extracted with RIPA buffer (Sigma-Aldrich, St. Louis, MO, USA), and the concentration of extracted protein was determined by BCA kit. The protein samples were mixed with the loading buffer and heated at 95 °C for 10 min. According to the target protein size, appropriate protein separation gels were configured for sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), and 25 μg of protein samples were loaded into each well. Proteins were subsequently transferred to polyvinylidene difluoride (PVDF) membranes. These membranes were then blocked with 5% BSA for 1 h at room temperature and subsequently incubated with primary antibodies overnight at 4 °C. Following primary antibody incubation, the PVDF membranes were washed four times with 1× Tris-buffered saline-tween (TBST) for 10 min per wash. They were then exposed to the appropriate secondary antibodies for 1 h at room temperature. Protein bands were visualized on a chemiluminescence imager using the ECL Western blotting substrate (Meilunbio, Dalian, China). Protein levels were quantified using Image J 1.53 analysis software, and each protein was normalized against β-actin levels. Details of antibodies are shown in Table 2.

The total protein of HK-2 was extracted with RIPA buffer (Beyotime, China) according to the manufacturer's instructions. Protein samples were subjected to 10% SDS-PAGE and then transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, USA). After blocking with 5% BSA (Solarbio, China), the membranes were incubated with corresponding primary antibodies: anti-Klotho (1:2000, Proteintech, USA), anti-pSmad3 (1:1000, ABclonal, China), anti-Smad3 (1:2000, Proteintech, USA), anti-PPARα (1:1000, Proteintech, USA), Anti-PGC1α (1:1000, ABclonal, China) and anti-GAPDH (1:10,000, Proteintech, USA). After incubation with appropriate secondary antibodies, the western blots were visualized using the ECL Western Blotting Substrate (Meilunbio, China). The signals were quantified with ImageJ software.