Anti pgc 1α

Epitrochlearis and soleus muscles were homogenized as previously described [21 (link)]. Primary antibodies were purchased as follows. From Cell Signaling Technology (Beverly, MA, USA); anti-AMPK-α, phospho-(p-)AMPK (Thr172), citrate synthase (CS), cytochrome C oxidase (COX) IV and SIRT1. From Merck (Temecula, CA, USA); anti-PGC-1α, monocarboxylate transporter (MCT) 1, MCT4 and MCT4. Anti-carnitine palmitoyltransferase (CPT) 1B, glucose transporter (GLUT) 4 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were obtained from Abcam (Tokyo, Japan), Biogenesis (Poole, United Kingdom) and Sigma-Aldrich (St. Louis, MO, USA), respectively. Equal protein concentrations were loaded in each lane and also visually confirmed by coomassie brilliant blue staining of the blot membrane. The phosphorylated AMPK abundance was normalized by AMPK-α abundance and other proteins were normalized by GAPDH abundance.

Immunoprecipitation was performed with muscle lysate of normal ICR mice as described [24 (link)]. The antibodies used are listed in Table S2.
Duolink in situ proximity ligation assay (PLA) was carried out according to the manufacturer’s instructions (Sigma-Aldrich, St. Louis, MO, USA). Briefly, antigen retrieval of deparaffinized 4 µm paraffin sections was carried out with 10 mM citrate buffer, pH 6. Frozen sections of WAT (25 µm) were fixed with 2% PFA. Muscle and WAT sections were permeabilized with 0.1% or 0.3% Triton-X100 for 30 or 15 min at RT, respectively. Sections were blocked with CAS-block (Invitrogen) for 1 h at RT and incubated O/N at +4 °C with primary antibodies diluted in ChemMate™ (DakoCytomation, Glostrup, Denmark). Primary antibodies used are listed in Table S2. Images were captured using Zeiss Axioplan2 microscope (Carl Zeiss Microscopy, Thornwood, NY, USA) and quantified with the Duolink Image Tool software (Olink Bioscience, Uppsala, Sweden).
Pull-down assay with WWE Affinity Resin Kit (Tulip Biolabs, West Point, PA, USA) was performed according to the manufacturer’s instruction. PARylated PGC-1α and TNKS1 were detected by immunoblotting the affinity precipitates with anti-PGC-1α (EMD Millipore) and anti-TNKS1/2 (Santa Cruz Biotechnology) IgGs.

The following primary antibodies were used for immunoblots: Anti-Hexokinase II (Cell Signaling Technology, 2867), Anti-GLUT1 (Cell Signaling Technology, 12939S), Anti-GLUT4 (Cell Signaling Technology, 2213S), Anti-CPT1β (Abcam, ab134988), Anti-VLCAD (Abcam, ab155138), Anti-Acadm (Santa Cruz, sc365108), Anti-PPARα (Cayman, 101710 and Novus Biologicals, N300-537), Anti-PGC-1α (Millipore, Ab3242), Anti-GAPDH (Cell Signaling Technology, 2118C), Anti-Tubulin (Cell Signaling Technology, 3873S). Secondary antibodies used were Anti-Rabbit IgG, HRP-link (Cell Signal Technology, 7074S) and Anti-Mouse IgG, HRP-link (Cell Signal Technology, 7076S). In all Western blot images shown in figures, individual lanes correspond to individual mice. Molecular weights (MW) are shown. The signal intensity of Western blots was quantified using ImageJ software and then normalized by the corresponding loading control (Tubulin or GAPDH).

HLE and Huh7 cells following different treatments were seeded in confocal dishes (NEST, VA, USA) and maintained in DMEM for 24 h. The cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100, and then incubated with primary antibodies overnight at 4 ˚C. Antibodies were used as follows: anti-ZNF281 (1:50, Santa Cruz, CA, USA), anti-TFAM (1:50, Cell Signaling Technology Inc., Danvers, MA, USA), anti-NRF1 (1:50, Cell Signaling Technology Inc., Danvers, MA, USA), anti-PGC-1α (1:50, Millipore, Billerica, MA, USA), anti-SDHB (1:200, Abcam, Cambridge, MA, USA), and anti-MT-CO2 (1:200, Abcam, Cambridge, MA, USA). The fluorescent secondary antibodies used were Alexa fluor (m) 594 goat A or Alexa fluor (R) 488 goat A (1:200, Invitrogen, NY, USA). Counter-staining of the nuclei was performed using 4′, 6-diamidino-2-phenylindole (DAPI). Samples were observed under a Leica DM5000 B fluorescent microscope and Leica SP5 confocal microscope (Leica Microsystems, Wetzlar, Germany).

Adult male flies aged 5–7 days were snap frozen and heads were separated into groups of 10 on dry ice. Protein extracts were prepared from the heads using extraction buffer 20 mM Hepes (pH 7.5), 100 mM KCl, 5% glycerol, 100 μM NA3VO4, 10 mM EDTA, 0.1% Triton X, 1 mM DTT, and (Phosphatase/protease inhibitors) 4X LDS (Invitrogen) and 10X Reducing Agent (Invitrogen) were added before samples were incubated at 70 °C for 10 min to denature and reduce. Samples were separated on a 4–12% Bis-Tris gel (Invitrogen) and transferred to a PVDF membrane (Immobilon-P, Millipore, St. Louis, MO). Enhanced chemiluminescence (SuperSignal West Pico; Thermo Scientific) was used for antibody detection. The following primary antibodies were used: anti-PGC-1α 1:1000 (Millipore, St. Louis, MO), anti-β-tubulin E7 1:20,000 (Developmental Studies Hybridoma Bank, Iowa City, IA). The relative intensity of PGC-1α to β-tubulin was determined using ImageJ (NIH).