Western Blot Analysis of Signaling Proteins

Western blotting analysis was applied to examine protein levels using antibodies recognizing Rho A (Abcam, Cambridge, MA, USA), ROCK1 (Abcam), ROCK2 (Abcam), myosin phosphatase-targeting subunit 1 (MYPT1) (Cell Signaling Technology (CST), Danvers, MA, USA), phosphorylated (p)-MYPT1 (CST), glucose transporter type 1, erythrocyte/brain (GLUT1) (Abcam), hexokinase 2 (HK2) (Abcam), pyruvate dehydrogenase kinase 1(PDK1) (Abcam), phosphofructokinase 1 (PFK1) (CST), lactate dehydrogenase A (LDHA) (Abcam), osteopontin (OPN) (Abcam), RUNX2 (Abcam), AMPK (Abcam), p-AMPK (Abcam), ubiquitination (Proteintech, Rosemont, IL, USA), Sodium Potassium ATPase (Abcam), and β-actin (Proteintech). Human aortic valve tissues and VICs were rinsed with cold 1× PBS, and then lysed on ice in radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime Biotech; 50 mM Tris, pH 7.4, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 1 mM sodium orthovanadate, 1 mM sodium fluoride, 1 mM Ethylenediaminetetraacetic acid (EDTA), and leupeptin) containing 1 mM phenylmethylsulfonyl fluoride (PMSF) for 15 min, followed by homogenization with 20 kHz ultrasonic lapping and centrifugation at 14,000 rpm at 4 °C in a refrigerated microcentrifuge for 15 min to extract total protein. Then, supernatants were stored and a bicinchoninic acid (BCA) Protein Assay Kit (Beyotime Biotech) was used to detect the total protein concentration to normalize the samples. Equal amounts of samples (30–45 μg/lane) were run on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels and then transferred to polyvinylidene difluoride (PVDF) membranes using a wet-transfer system. The membranes were blocked using QuickBlock Blocking Buffer (Beyotime Biotech) at room temperature for 20 min, and then incubated with primary antibodies at 4 °C overnight. After three washes with Tris-buffered saline-Tween-20 (TBST) (Servicebio, Wuhan, China), we incubated membranes with the corresponding secondary antibodies coupled with horseradish peroxidase (HRP) for 90 min on shakers. After three washes with TBST, enhanced chemiluminescence (ECL) signals (Amersham Biosciences, Piscataway, NJ, USA) were detected using an ECL kit (Millipore, Billerica, MA, USA) with Imaging system (Thermo Fisher Scientific). Densitometric quantification was performed using Image J software.