Matrix metalloproteinase 2 mmp2

The total protein was extracted with a RIPA lysis buffer, and its concentration was determined with a BCA kit (Solarbio). The polyacrylamide gel was prepared in advance, and its concentration was determined by the size of the protein to be detected. The protein sample was loaded into a polyacrylamide gel, followed by electrophoresis for 2–3 h. Afterward, the protein was transferred onto a polyvinylidene fluoride membrane (Millipore, Billerica, MA, USA), and skim milk was used to block the heterogenetic antigens. Subsequently, the membrane loaded with protein was incubated with primary antibody at 4°C in the dark overnight. After rinsing with TBST buffer, the membrane was incubated with a secondary antibody labeled with HRP, and reacted with ECL reagent (Solarbio) for several minutes, followed by signal exposure in the dark. The antibody information was shown in the following: RASIP1 (1:1000; Affinity, Changzhou, Jiangsu, China), cyclin E1 (1:1000; Affinity), cyclin B1 (1:1000; Affinity), cyclin D1 (1:1000; Affinity), p27 (1:1000; Affinity), matrix metalloproteinase 2 (MMP2) (1:2000; Novus Biologicals, Littleton, CO, USA), MMP9 (1:1000; Affinity), cleaved caspase-3 (1:1000; Affinity), Bax (1:1000; Affinity), Bad (1:1000; Affinity), Bcl-xl (1:1000; Affinity), FOXO3 (1:500; Affinity), GAPDH (1:10000; Affinity).

Briefly, protein was extracted from the aorta using radioimmunoprecipitation assay (RIPA) buffer (Beyotime, China) and quantified using a BCA Protein Kit (CWBio, China). Proteins were separated using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes. The PVDF membranes were blocked with 1% bovine serum albumin (BSA) for 1 h, and then incubated with primary antibodies against p-AKT (Cell Signaling Technology, USA), AKT (Cell Signaling Technology, USA), p-mTOR (Cell Signaling Technology, USA), mTOR (Cell Signaling Technology, USA), p62 (Cell Signaling Technology, USA), LC3 (Sigma-Aldrich, USA), SM22α (Abcam, UK), α-SMA (Abcam, UK), myocardin (Abcam, UK), OPN (Abcam, UK), matrix metalloproteinase-2 (MMP-2; Novus, USA), matrix metalloproteinase-9 (MMP-9; Abcam, UK), p65 (Cell Signaling Technology, USA), TNF-α (Abcam, UK), and β-actin (Abcam, UK), overnight at 4°C. Next, membranes were washed three times with tris buffered saline tween (TBST) and incubated for 1 h with secondary antibodies (Abcam, UK). Proteins were detected using enhanced chemiluminescent reagents (Thermo Fisher Scientific, USA) and quantified using ImageJ software (NIH, USA).