Anti nrf1

The protein expression levels of peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC-1α), peroxisome proliferator-activated receptor alpha (PPARα), nuclear respiratory factor 1 (NRF1), and mitochondrial transcription factor A (mtTFA) were measured by Western blot (Chen et al., 2018 (link)). The specific methods and steps are shown in Part 2 of the Supplementary Materials. The following antibodies were used: anti-PGC-1α (batch number: ab54481), anti-PPARα (batch number: ab8934), anti-NRF1 (batch number: ab175932), and anti-mtTFA (mitochondrial marker, batch number: ab131607) were provided by Abcam (Cambridge, UK); anti-β-actin (batch number: TA-09) was supplied by Beijing Zhongshan Jinqiao Biotechnology Co., Ltd. (Beijing, China). The mRNA expression levels of PGC-1α, PPARα, NRF1, and mtTFA were measured by real-time PCR (RT-PCR) (Mocker et al., 2019 (link)). The specific methods and steps are shown in Part 3 of the Supplementary Materials. Mitochondrial DNA (mtDNA) was detected using the PCR-fluorescent probe method (Liu et al., 2016 (link)). The specific methods and steps are shown in Part 4 of the Supplementary Materials.

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).

Frozen hearts, livers and kidneys were homogenized in modified RIPA buffer containing 50 mM Tris/HCl, 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxychoilate, 0.1% SDS, 1 mM EDTA, 10 mM sodium fluoride, Protease inhibitor cocktail (Roche Life Sciences, Mannheim, Germany) and Phosphatase inhibitor cocktail 2 and 3 (Sigma-Aldrich, Taufkirchen, Germany), pH 7.5 using an Ultra-Turrax T10 basic homogenizer. Samples were separated by 10% SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes at 250 mA for 2 h and incubated with the following primary antibodies: anti-insulin receptor (1:1000, Cell Signaling Technologies, 3025), anti-NRF1 (1:2000, Abcam, ab175932). Anti-Rabbit IgG (H+L) Fab2 Alexa Fluor (1:5000; Cell Signaling, 4414) and anti-Mouse IgG (H+L) Fab2 Alexa Fluor (1:10000; Cell Signaling, 4408) served as secondary antibody. Detection and quantification of fluorescent bands was performed using the Bio-Rad Western Blot Imager. Loading control was performed using anti-alpha-tubulin (1:2000, Sigma-Aldrich, T9026). For blots investigating levels of MTHFD1L, ATP synthase β and alpha-tubulin performed using isolated mitochondria, the following primary antibodies were used: Anti-MTHFD1L (1:1000, Novus Biologicals NBP2-37864), anti-ATP synthase β (1:2000, BD Biosciences, 612519), anti-alpha-tubulin (1:2000, Sigma-Aldrich, T9026).