Anti pgc 1α

Monoclonal anti-actin antibody, Prussian Blue, pantothenate, pantethine, vitamin E, L-carnitine, thiamine, Luperox^® and trypsin were purchased from Sigma Chemical Co. (St. Louis, MO). Anti-mitochondrial acyl carrier protein (mtACP), anti-NF-Y, anti-FOXN4 and anti-hnRNPA/B were purchased from Invitrogen/Molecular Probes (Eugene, OR). Anti-phospho-PGC1α was purchased from RD systems. NFS1 antibody and Omega 3 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-PANK2, anti-PGC1α, complex 1 activity kit and PDH activity kit were purchased from Abcam (Cambridge, UK). Anti-TFAM was purchased from Cell Signaling. BODIPY^® 581/591 C11 was purchased from Thermo-Fisher (Waltham, MA). A cocktail of protease inhibitors (complete cocktail) was purchased from Boehringer Mannheim (Indianapolis, IN). The Immun Star HRP substrate kit was from Bio-Rad Laboratories Inc. (Hercules, CA).

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.

Protein extracts were obtained from kidneys using T-PER Mammalian Protein Extraction Reagent (Pierce, Thermo Scientific, Rockford, USA), as indicated by the manufacturer, in the presence of a cocktail of protease and phosphatase inhibitors (Sigma-Aldrich, Milan, Italy). Protein content was determined with bicinchoninic acid protein assays (BCA, Pierce, Euroclone, Milan, Italy). An appropriate amount of protein was run on SDS-PAGE under reducing conditions for immunoblotting. The separated proteins were then semi-dry transferred to a nitrocellulose membrane (Bio-Rad Laboratories) and proteins of interest were revealed with specific antibodies: anti-p-S6 (Ser235/236), anti-S6, anti-p-AKT (Ser473), anti-AKT, anti-p-eNOS (Ser1177), anti-eNOS, anti-COX IV, anti-Cyt c, and anti-Grp78 (all from Cell Signaling, Euroclone, Milan, Italy); anti-SIRT1, anti-Grp75, and anti-Bcl-2 (all from Santa Cruz); and anti-PGC-1α (Abcam, Cambridge, UK) each at 1:1,000 dilution. Anti-β-Actin (1:10,000; Cell Signaling) and anti-Vinculin (1:10,000; Sigma-Aldrich) were used as loading controls. Immunostaining was detected using horseradish peroxidase conjugated anti-rabbit or anti-mouse immunoglobulin for 1 h at room temperature (Tedesco et al. 2010 (link)). Amounts of each protein were measured using SuperSignal Substrate (Pierce) and densitometrically quantified with an IMAGEJ software image analyzer.

Proteins were extracted with lysis buffer (50 mM HEPES, 100 mM NaF, 10 mM EDTA, 50 mM Na pyrophosphate, 1% Triton at pH 7.4, and 10 mM Na Orthovanadate) for 15 min at 4 °C as previously reported [17 (link)]. The total protein samples (25 µg) were subjected to 9% SDS PAGE, transferred onto an activated polyvinylidene difluoride membrane (Immobilon P, Millipore, Denmark), and incubated overnight at 4 °C with primary antibodies followed by peroxidase-conjugated secondary antibodies. The signal was detected by Super Signal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, San Jose, CA, USA). Images were obtained using a densitometer (Bio-Rad Laboratories, Hercules, CA, USA) and resolved quantitatively. Each band level was standardized with respect to the β-actin control. Anti-asprosin (AdipoGen, Seoul, Korea), anti-PKA, anti-GLUT4, anti-SOD1, anti-SOD2, anti-β-actin antibodies (Santa Cruz, CA, USA), anti-CRBN (Sigma-Aldrich, Louis, MO, USA), anti-AMPK, anti-Akt, anti-p38MAPK, anti-TGF-β (Cell Signaling Technology, Beverly, MA, USA), anti-FNDC5, anti-PGC-1α, and anti-UCP3 antibody (Abcam, Cambridge, MA, USA) were used.

The instruments and reagents used in these experiments were as follows: a 7.0T animal magnetic resonance instrument (Bruker, Germany); novel object recognition and test system (Boster Bioengineering, China); Y labyrinth video analysis system (Shanghai Xinruan Information Technology, China); high-fat feed (21% fat, 0.15% cholesterol, Jiangsu Medisen Biomedicine, China); isoflurane (Shenzhen Reward, China); immunohistochemistry kit and DAB staining kit (Boster Bioengineering, China); and haematoxylin staining solution and eosin staining solution (Beijing Solebao Technology, China). The primary antibodies in this study were as follows: anti-SIRT1 (Proteintech, USA), anti-TNF-α (Proteintech, USA), anti-β-actin (Proteintech, USA), anti-IBA1 (Proteintech, USA), anti-GFAP (Proteintech, USA), anti-PGC-1α (Abcam, USA), anti-BDNF (Abcam, USA), anti-IL-1β (Abcam, USA), NF-κBp65 antibody (Cell Signaling Technology, USA), goat anti-mouse/anti-rabbit secondary antibody (Proteintech, USA), and antibody diluent (Beijing Biyuntian, China).

Whole protein lysates from mouse intestinal tissues were prepared using RIPA lysis buffer (Thermo Fisher Scientific, Rockford, IL, USA). Samples were loaded into 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gels (BIO-RAD, Hercules, CA, USA), transferred to polyvinylidene difluoride membranes (Amersham Biosciences, Buckinghamshire, UK) and incubated with primary anti-K8 antibody (Troma-1, MilliporeSigma, USA,1:500), antiPGC-1α (Abcam, Cambridge, MA, USA, 1:500), anti-phospho-MLKL (p-MLKL) (CST, 1:500), anti-AKT (CST, 1:500), anti-cleaved caspase 3 (CST, 1:500), anti-GAPDH (CST, 1:1,000) and anti-cleaved-K18 (AnaSpec, Inc., Fremont, CA, USA, 1:1,000) overnight at 4°C. Membranes were incubated with secondary monoclonal goat anti-rat antibody (Immunoreagents, Inc., Raleigh, NC, USA, 1:1,000) or goat anti-rabbit antibody (CST, 1:1,000) at room temperature for 1 h. Protein bands were visualized using an ECL detection kit (Thermo Fisher Scientific, Rockford, IL, USA) according to the manufacture's protocol.