Anti pgc 1α

For immunoblotting, antibodies used in this study were purchased from EMD Millipore (Oakville, Canada), Sigma Aldrich (Oakville, Canada), Proteintech (Illinois, USA), and Cell Signaling Technology (Whitby, Ontario). The following primary antibody was used from EMD Millipore (Oakville, Canada): anti-androgen receptor (Millipore Cat# 06-680, 1:1,000). The following primary antibodies were used from Sigma Aldrich (Oakville, Canada): anti-fast skeletal myosin (M4267, 1:15,000), anti-slow skeletal myosin (Sigma-Aldrich Cat# M8421, 1:10,000), and anti-beta actin (Sigma-Aldrich Cat# A2066, 1:10,000). The following primary antibodies were used from Proteintech (Illinois, USA): anti-PGC1α (Proteintech Cat# 66369-1-Ig, 1:5,000), anti-NRF-2 (Proteintech Cat# 16396-1-AP, 1:500), and anti-TFAM (Proteintech Cat# 22586-1-AP, 1:1,000). The following HRP-conjugated secondary antibodies were used from Cell Signaling Technology (Whitby, Ontario): goat anti-rabbit IgG, HRP-linked (Cell Signaling Technology Cat# 7074, 1:5,000) and horse anti-mouse IgG, HRP-linked (Cell Signaling Technology Cat# 7076, 1:5,000).

C2C12 myoblasts were transfected with NT siRNA or Tet3 siRNA as described above for 48 h, followed by addition of cycloheximide (CHX) (Cell Signaling Technology, MA, USA 2112) at a final concentration of 50 μg/ml. Proteins were isolated at 0, 15, 30 and 45 min later and analysed by western blotting using anti-PGC-1α (dilution 1:1000; Proteintech, IL, USA, 66369-1-Ig) and horseradish peroxide (HRP)-conjugated anti-GAPDH (dilution 1:5000; Proteintech, HRP-60004).

The total proteins were extracted from cell samples using RIPA lysate buffer containing PMSF and phosphatase inhibitor. The Pierce BCA protein assay kit (Thermo Fisher Scientific, Germany) was used to quantify proteins. Equal amounts of proteins were separated by SDS-PAGE, using 7% polyacrylamide gels and then transferred onto PVDF membranes (Millipore, USA). The membranes were blocked with 1 × TBS buffer containing 5% BSA and 0.05% Tween 20 for 3 h, incubated with primary antibodies at appropriate dilutions for 2 h and subsequently incubated with peroxidase-conjugated goat-anti-rabbit IgG (1:5000 dilutions) for 1 h. Immunoreactive proteins were visualized by using DAB staining (Beyotime, China). Primary antibodies used in this study included anti-mTOR (7C10), anti-p-mTOR (ser2448) (Cell Signaling Technology, Danvers, MA), anti-SIRT3, anti-GLS1, anti-GDH, anti-IDH2, anti-PGC-1α, anti-α-KGDH (Proteintech, USA), and anti-β-actin (Abcam, USA).

Tissues and cells were lysed with RIPA buffer containing Halt Protease/Phosphatase inhibitors (Thermo Fisher Scientifics, Pittsburgh, PA). The primary antibodies used in this study were as follows: anti-phospho (Ser563)-HSL (#4139), anti-phospho (Ser660)-HSL (#4126), anti-HSL (#4107), anti-pPKA Substrate (#9621), and anti-phospho (Ser133)-CREB (#9198) (all from Cell Signaling Technology); anti-SLC22A3 antibody (#ab191446), anti-ATP5A (#ab176569), anti-COX7B (#ab140629), anti-UQCRH (#ab134949), anti-UQCRB (#ab190360), and anti-beta Actin (ab8226) (all from Abcam); anti-UCP1 (#GTX112784) (Genetex); and anti-PGC1α (#20658) (Proteintech Group Inc).

Male Sprague-Dawley (SD) rats were purchased from Sina-British Sippr/Bk Lab Animal Ltd. (Shanghai, China). Tetramethylpyrazine (TMP), Crocin, Ferulic acid and Chlorogenic acid, acetylcholine, phenylephrine (PE) and D-glucose were purchased from Sigma-Aldrich Co. (St Louis, Missouri, USA). Dulbecco's modified Eagle's medium (DMEM) supplemented with 5.6 mmol/L glucose was obtained from Hclone (Logan, UT, USA). DMEM without red phenol, fetal bovine serum (FBS) and Trizol were purchased from Invitrogen (Carlsbad, CA, USA). CM-H₂DCFDA, DAF-FM diacetate and MitoSOX™ Red mitochondrial superoxide indicators were obtained from Invitrogen (Carlsbad, CA, USA). JC-1 indicator was bought from Molecular Probes (Eugene, OR, USA). Antibodies including anti-complex III, anti-PGC-1α, anti-SIRT1 and anti-β-actin were purchased from Proteintech (ProteinTech Group, Chicago, IL, USA). Primers for PGC-1α, NRF-1, TFAM, SIRT1 and 18s were provided by Sangon Biotech CO. Ltd. (Shanghai).

We collected hippocampal proteins in a radioimmunoprecipitation assay buffer containing phenylmethylsulfonyl fluoride (100:1). Protein concentrations were determined with a bicinchoninic acid assay kit (Solarbo, Beijing, China). Proteins were treated with a sodium dodecyl sulfate- (SDS-) loading buffer after quantitative assessment, subjected to 12% SDS polyacrylamide gel electrophoresis, and then transferred onto polyvinylidene difluoride membranes (5 μl for each sample, 80 V, 100 min). After blocking with 5% dried skim milk for 1 h, the membranes were incubated with anti-SIRT1 (1:2400), anti-PGC-1α (1:2000), anti-NRF1 (1:3000), and anti-β actin (1:10,000) antibodies (Proteintech, Rosemont, IL) at room temperature for 1 h. They were then incubated with IRDye secondary antibodies (1:10,000; ZSGB-BIO, Beijing, China) for 1 h at room temperature. After the membranes were washed five times with tris-buffered saline-Tween 20 for 25 min, an ECL Prime western blotting detection reagent (GE Healthcare, Chicago, IL) was used to expose protein bands. Protein bands were visualised with a FluorChem M gel image analysis system (Alpha Innotech).

Gastrocnemius muscle tissues or C2C12 myotubes were lysed in RIPA enhancer buffer (Beyotime, Shanghai, China, P0013B) and supplemented with protease inhibitor cocktail (Beyotime, Shanghai, China, P1005, 1:100). The lysates/proteins were loaded at 20 μg per lane, after electrophoresis and transfer, the PVDF membrane was incubated with primary antibody, the following primary antibodies: anti-HIF-α (CST, Danvers, MA, USA, #36169, 1:1000), anti-FNDC5 (Abcam, Waltham, MA, USA, ab174833; 1:2000), anti-β-tubulin (Abcam, Waltham, MA, USA, ab6046, 1:2000), anti-GAPDH (Abcam, Waltham, MA, USA, ab8245, 1:1000) and anti-PGC-1α (Proteintech, Wuhan, China20658-1-AP, 1:2000) were incubated overnight at 4 ℃. The HRP-coupled secondary antibody (Immunoway, Jiangsu, China, RS0001, 1:10,000) was incubated for 1 h at room temperature (RT). Protein bands were displayed via clarity^TM western ECL substrate (BIO-RAD, Hercules, CA, USA #170-5060) by using Chemiluminescence Apparatus (Tanon, Shanghai, China, T5200).