Goat anti rabbit fluorescent secondary antibody

The restriction endonuclease BsmB I and T4 DNA ligase were purchased from NEB, Inc. (Ipswich, MA, USA). The gel recovery kit, PrimeScript RT reagent Kit with gDNA Eraser reverse transcription kit, SYBR Premix ExTaq RT-PCR kit and competent cells were purchased from TaKaRa Bio Japan, Inc. (Tokyo, Japan). The plasmid isolation kit was purchased from Omega Bio-tek, Inc. (Norcross, GA, USA). Rabbit GOT1 primary antibodies (bs-3977R), rabbit MDH1 primary antibodies (bs-3996R) and rabbit β-actin primary antibodies (bs-0061R) were purchased from Bioss, Inc. (Woburn, MA, USA). Goat anti-rabbit fluorescent secondary antibodies were purchased from LI-COR Biosciences, Inc. (Lincoln, NE, USA). Penicillin–streptomycin and NADP/NADPH assay kits were purchased from Solarbio Science & Technology Co., Ltd. (Beijing, China). Low-glucose Dulbecco’s modified eagle medium (DMEM) and foetal bovine serum (FBS) were purchased from Gibco, Inc. (San Diego, CA, USA).

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.