Cpt1b

Protein levels were measured by WB with β-actin as the loading control. Protein was extracted from atria samples or cells and quantitated by a BCA assay. Equal amount (10–30 μg) of protein was loaded and separated by SDS-PAGE using 10 or 12% acrylamide gradients, and later transferred to nitrocellulose membranes. The membranes were blocked with 5% skim milk and incubated with antibodies against total-AMPK (1:1,000; Cell Signaling Technology/CST, Massachusetts, United States), CD36 (1:1,000; Abcam), collagen Ⅰ (1:1,000; Abcam), collagen Ⅲ (1:1,000; Abcam), connexin-43 (1:200; Invitrogen), CPT1B (1:1,000; Proteintech), GLUT4 (1:500; Proteintech), NFκB (1:1,000; Proteintech), NRF2 (1:1,000; Proteintech), phoso-AMPK (Thr172; 1:1,000; CST), phoso-Akt (Ser473; 1:1,000; CST), phoso-NFκB (1:1,000; CST), PGC1α (1:1,000; Proteintech), SOD2 (1:1,000; Proteintech), TGF-β (1:1,000; Abcam), α-SMA (1:1,000; CST), β-actin (1:5,000; Proteintech). Protein levels were quantified as the intensity of bands using ImageJ software (NIH systems) and standardized to β-actin. Abbreviations are fully illustrated in corresponding figure legends.

The muscle tissue (~100 mg) was homogenized with a steel bead in 1 ml of cold RIPA buffer containing 1X Proteinase Inhibitor Mix (complete^™ Protease Inhibitor Cocktail, Roche, Cat. no.11 697 498 001), 1X PhosStop (Roche, Mannheim Germany, Cat. no.04 906 845 001) using a TissueLyser II instrument (Qiagen) set at 30 strokes/s for 2–4 min. Based on protein quantification results, all samples were adjusted to the final concentration of 2 μg/ul and heat-denatured for 5 min at 99 °C in 2X Laemmli buffer. Proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes. The membranes were incubated in Odyssey blocking solution for 1 h. Total proteins were detected by probing the membranes with appropriate primary antibodies overnight at 4 °C. The following antibodies were used: Chkα (1:1000, Abcam Cat#ab88053), Pparα (1:1000, Abcam, Cat#Ab24509), Pparb (1:1000, Biorad, Cat#AHP1272), Cpt1b (1:1000, Proteintech®, Cat#22170-1-AP), Chkβ (1:250, Santa Cruz, Cat#398957), GAPDH (1:1000, Cell signaling, Cat#2118), Pparγ (1:500, Santa Cruz, Cat# sc-7273). Proteins were visualized with goat anti-rabbit IRDye-800- or −680-secondary antibodies (1:20,000, LI-COR Biosciences, Cat#926-32211 and Cat#926-68071) or anti-mouse m-IgGκ BP-CFL 790 (Santa Cruz, Cat. no.sc-516181) using an Odyssey imaging system and band density were evaluated using FIJI (NIH).