Anti fndc5

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 total protein was separated by SDS‐PAGE and transferred to PVDF membranes. The membranes were incubated with the corresponding primary antibodies, namely anti‐phosphorylated (p)‐AMPK (Thr172; 1:1000; CST), anti‐AMPK (1:1000, CST), anti‐SIRT‐1 (1:1000, Abcam), anti‐p‐FOXO‐3a (Ser253; 1:500; Zen‐Bio Science), anti‐FOXO‐3a (1:1000, GeneTex), anti‐Atrogin‐1 (1:1000, Abcam), anti‐MuRF‐1 (1:1000, Proteintech), anti‐p‐IRS‐1 (Ser307; 1:500; Zen‐Bio Science), anti‐IRS‐1 (1:1000, Zen‐Bio Science), anti‐GLUT‐4 (1:500, Proteintech), anti‐MHC (1:200, Santa Cruz), anti‐MyoD (1:200, Santa Cruz), anti‐Myog (1:200, Santacruz), anti‐PGC‐1α (1:1000, Abcam), anti‐FNDC‐5 (1:1000, Abcam) and anti‐GAPDH (1:5000, Proteintech) overnight at 4°C. Then, the membranes were incubated with the secondary antibodies for 1 h at 37°C. After treatment with an enhanced chemiluminescence substrate kit (Advansta), the protein bands were detected using Fusion software.

Here, 20 µg of protein from the myoblast cell cultures were solubilized with a lysis buffer (50 mM Tris (Tris(hydroxymethyl)aminomethane)-HCl (pH 8.0), 150 mM HCl, 5 mM ethylenediaminetetraacetic acid, 1% NP40 and 1 mM phenylmethyl sulfonyl fluoride). The protein concentration was measured using a DC™ Protein Assay (Bio-Rad Laboratories, Hercules, CA, USA). The cell lysates were subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to nitrocellulose membranes (Sigma-Aldrich, St. Louis, MO, USA). The blots were incubated overnight at 4 °C using primary antibody anti-Fndc5, anti-Sirt1, anti-Pgc1α, and β-Actin (Abcam, Cambridge, UK). Subsequently, the membranes were incubated for one hour at room temperature with IRDye-labeled secondary antibodies (680/800 CW) (LI-COR Corp., Lincoln, NE, USA). The Odyssey infrared imaging system (LI-COR Corp., Lincoln, NE, USA) was utilized for immunodetection. All data were normalized to loading controls.