Rabbit anti pparα

Primary antibodies: rabbit anti-PPARα, Abcam (Cambridge, MA), 1:50; rabbit anti-PPARβ/δ, Pierce (Rockford, IL), 1:100; rabbit anti-PPARγ, Abcam, 1:20; mouse anti- neuronal nuclei-neuron specific nuclear protein (NeuN), Millipore (Billerica, MA), 1:500; mouse anti-glial fibrillary protein (GFAP), NeuroMab, 1:300; goat anti-ionized calcium-binding adapter 1 (Iba1), Abcam, 1:300. Secondary antibodies: goat anti-rabbit 488, donkey anti-rabbit 488, goat anti-mouse 594 or donkey anti-goat 568, Invitrogen (Grand Island, NY), 1:1,000. For complete list of antibodies see Supplemental Table S2.

Total protein was extracted from liver, muscle, and kidney tissues using radioactive immunoprecipitation (RIPA) lysates. Approximately 50–80 μg of protein was run on a discontinuous SDS-PAGE gel and transferred to a nitrocellulose membrane. The membranes were blocked with 5% skim milk in TBST containing 0.05% tween for 2 h and were then incubated with the following primary antibodies overnight at 4°C: (1) rabbit anti-ADPNR-1 (1:500), rabbit anti-ADPNR-2 (1:300), and rabbit anti-TGFβ-1 (1:200) (Santa Cruz Biotech, USA); (2) rabbit anti-PPAR-α (1:200), rabbit anti-G-6-P (1:500), and mouse anti-GLUT-4 (1:500) (Abcam, USA); (3) rabbit anti-AMPKα (1:1500), rabbit anti-Phospho-AMPKα (1:1000) (Cell Signaling Technology, USA); and (4) rabbit anti-GADPH (1:2000, Bioworld Tech). The membrane was washed and incubated at room temperature for 2 h in the dark with goat anti-rabbit fluorescent secondary antibodies (Li-COR Bioscience, USA). The NC membranes were scanned with an Odyssey infrared fluorescence scanner (Li-COR Bioscience, USA). The optical density (OD) of the signals was quantified and expressed as the ratio of OD of the tested proteins (PPAR-α, G-6-P, GLUT-4, ADPNR-1, ADPNR-2, and TGFβ-1) to that of GADPH. The protein expression of AMPK phosphorylation parameters was expressed as the pAMPK and AMPK striped gray value ratio.