Chemstudio system

Liver samples (100 mg) were homogenized with 400 μL of RIPA lysis buffer, containing 1% phenylmethylsulfonyl fluoride (PMSF) and 1% phosphatase inhibitor cocktail and then homogenized at 4°C for 1 min. Following homogenization, the samples were centrifuged at 12,000 g at 4°C for 30 min. The supernatant was then isolated to determine the protein concentration using a BCA protein assay kit. As per our previously described method (Han et al., 2023 (link); Zhang et al., 2022 (link)), the protein was loaded and separated through 15% sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE), and then transferred onto a polyvinylidene difluoride (PVDF) membrane. The membrane was blocked for 1 h at 25°C and then incubated overnight at 4°C with a primary antibody targeting either ESR1 or NR3C1. GAPDH served as the housekeeping protein. Following a TBST (Tris‐buffered saline with 0.1% Tween‐20) wash, the membranes were incubated with a secondary antibody for 1 h at room temperature. The membranes were subsequently treated with ECL solution and imaged using the ChemStudio system (Analytik Jena, Jena, Germany). Finally, the protein bands were semiquantitatively assessed using ImageJ software and normalized to β‐actin density.

Genomic DNA from human leukocytes or renal mouse tissue was extracted using a Quick-DNA™ Miniprep Plus Kit (D4068, ZYMO RESEARCH Corp.). An EZ DNA Methylation-Gold™ Kit (D5006, ZYMO RESEARCH Corp.) was used to convert unmethylated cytosines to uracil, so that the DNA could be analyzed by PCR amplification. Methylated Klotho, Unmethylated Klotho, and Input were detected using ZymoTaq™ PreMix (E2004, ZYMO RESEARCH Corp.; Yin et al., 2017 (link)). Human Klotho promoter (−315/−99) and Mouse Klotho promoter (+497/+685) were analyzed by methylation-specific PCR (MSP), because these positions were rich in CpG islands near transcription starting site (Sun et al., 2012 (link); Yin et al., 2017 (link)). Primers are listed in Supplementary Table 1 in accordance with the literature (Sun et al., 2012 (link); Yin et al., 2017 (link)). PCR products were analyzed by 1.2% (w/v) agarose gel electrophoresis and densitometry was conducted using a ChemStudio system (Analytik Jena AG, Germany). The quantification is presented as the ratio of methylated or unmethylated PCR products to the total PCR products.