Cells were lysed by a radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime) supplemented with phenylmethylsulfonyl fluoride, (PMSF) (Santa Cruz). The protein was extracted using a Total Protein Extraction kit (Beyotime) according to the manufacturer’s instruction. The doses of protein were examined with the bicinchoninic acid (BCA) protein assay kit (Beyotime). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to separate the proteins, which was then transferred to polyvinylidene difluoride (PVDF) membranes.
Primary antibodies used included antibodies to the proteins, SIRT1 (1: 4000)(Abcam)), AMPK (1: 2000) (Cell Signaling Tech), phosphorylated (p-AMPK) (1: 2000) (Cell Signaling Technology), mechanistic target of rapamycin (mTOR) (1: 4000) (Abcam), phosphorylated mTOR (p-mTOR) (1: 4000) (Abcam), cleaved caspase-3 (c-caspase-3) (1: 2500) (AbCam), active matrix metalloproteinase-2 (MMP-2) (1: 4000) (Abcam), active MMP-9 (1: 2000) (Abcam) and GAPDH (1: 5000) (Abcam) for 8 hours at 4°C. After washing, the membranes were further incubated with horseradish-peroxidase (HRP)-conjugated secondary antibodies (Cell Signaling Technology). Luminol enhancer reagent (Millipore) was used to develop the membranes, which were visualized on X-ray films. The intensity of the Western blots was analyzed by ImageJ software.
Primary antibodies used included antibodies to the proteins, SIRT1 (1: 4000)(Abcam)), AMPK (1: 2000) (Cell Signaling Tech), phosphorylated (p-AMPK) (1: 2000) (Cell Signaling Technology), mechanistic target of rapamycin (mTOR) (1: 4000) (Abcam), phosphorylated mTOR (p-mTOR) (1: 4000) (Abcam), cleaved caspase-3 (c-caspase-3) (1: 2500) (AbCam), active matrix metalloproteinase-2 (MMP-2) (1: 4000) (Abcam), active MMP-9 (1: 2000) (Abcam) and GAPDH (1: 5000) (Abcam) for 8 hours at 4°C. After washing, the membranes were further incubated with horseradish-peroxidase (HRP)-conjugated secondary antibodies (Cell Signaling Technology). Luminol enhancer reagent (Millipore) was used to develop the membranes, which were visualized on X-ray films. The intensity of the Western blots was analyzed by ImageJ software.