Anti errα

Following lysis with radioimmunoprecipitation assay buffer (MilliporeSigma) supplemented with protease inhibitors (Roche Diagnostics), equivalent proteins (20 μg) were separated by 10% SDS-PAGE. Subsequently, proteins were transferred to a PVDF membrane (MilliporeSigma) and were then blocked with 5% skim milk in PBS-Tween 20. The membrane was incubated with the following primary antibodies (dilution, 1:1000) at 4 °C overnight: Anti- GAPDH (cat. no. ab181602; Abcam), anti-ERRα (cat. no. sc‐65718; Santa Cruz Biotechnology, Inc.) and anti-IGF2BP1 (cat. no. 22803-1-AP; ProteinTech Group, Inc.). Following washing to remove primary antibodies, the membranes were incubated with the corresponding secondary horseradish peroxidase (HRP)-conjugated IgG antibody (dilution, 1:5000) at room temperature for 90 min. Enhanced chemiluminescence (ECL; Beyotime Institute of Biotechnology) was used to visualize the protein bands with the BioImaging System (GE Healthcare). The expression levels of ERRα and IGF2BP1 were normalized to those of GAPDH.

Myocardium tissue was harvested for Western blot as described previously [23 (link)]. Cells of each group were harvested at appropriate time. Cells were washed three times with PBS and collected after ice-cold lysis buffer digestion. Protein lysates were separated on 10% SDS-PAGE gels and transferred onto nitrocellulose (NC) membrane. Membranes were blocked with 5% milk in 1 × TBS-Tween-20 buffer and incubated overnight at 4°C with primary antibodies. Then, membranes were washed in Tris-buffered saline with Tween, followed by incubation with the corresponding secondary antibodies at room temperature for 1 h. The blots were developed using an enhanced chemiluminescence kit (Millipore, Billerica, MA, USA) and visualized with UVP Bio-Imaging Systems. Blot densities were analyzed using ImageJ Software (National Institutes of Health, Bethesda, MD).
Primary antibodies are the following: anti-SIRT1, anti-IRS2, anti-P-Akt S473, anti-P-Akt T308, anti-t-Akt, anti-acetylated protein, anti-PGC-1α, anti-NRF1, anti-NRF2, anti-ERR-α, anti-TFAM, anti-GAPDH, and anti-β-actin (all from Abcam, Cambridge, MA, USA). Secondary antibodies are the following: horseradish peroxidase-conjugated goat anti-rabbit and goat anti-rat (from Zhongshan Biotechnology Co. Ltd., Beijing, China).

After the intervention, the rats were anesthetized with intraperitoneal injection of 10% chloral hydrate (3 mL/kg). The kidneys were flushed with saline after the abdominal cavities were open, fixed with neutral formalin for 30–50 min, embedded in paraffin, and sectioned at 5-µm thickness. Paraffin sections were dewaxed and hydrated, and then antigen was mixed with EDTA antigen retrieval buffer (pH 9.0). Three percent hydrogen peroxide was used to block the activity of endogenous peroxidase. The sections were dehydrated, blocked in 3% bovine serum albumin (BSA) for 30 min, and then incubated with anti-ERRα (1:1000; Abcam, Cambridge, UK). Sections were flatted on a humid chamber at 4 °C overnight, and then washed three times in phosphate buffered saline (PBS, pH 7.4) for 5 min each. Secondary antibody horseradish peroxidase (HRP labeled) were added and incubated with the sections for 50 min. Freshly prepared solution of diaminobenzidine (DAB) (K5007, DAKO, Glostrup, Denmark) was added and then rinsed with tap water to terminate coloration. Sections were stained with hematoxylin for 3 min, rinsed with tap water and examined under a microscope after dehydration.