Ge image quant las4000 mini system

Western Blot Analysis was performed to detect SM α-actin, SM-MHC, C-Myb, BCL-2, Bax, β-tubulin, and β-actin protein levels. In brief, total proteins were extracted from cultured cells and measured using a BCA Protein Assay kit (Thermo, USA). Equal amounts of protein were subjected to 6% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA, USA). The membranes were blocked with 5% BSA for 1 h, followed by incubation with primary antibodies overnight at 4°C. The membranes were incubated with HRP-conjugated secondary antibody (A00098, GenScript, USA). The bands were imaged and analyzed on a GE Image-Quant LAS4000 mini system (GE Healthcare, USA). The primary antibodies used in the study were displayed as follows: anti-SM α-actin (ab5694, Abcam, UK), anti-SM-MHC (ab124679, Abcam, UK), anti-C-Myb (NBP1-80306, Novus, USA), anti-BCL-2 (NB100-92142, Novus, USA), anti-Bax (#2772, Cell Signaling Technology, USA), anti-β-tubulin (#2128, Cell Signaling Technology, USA), and anti-β-actin (#4970, Cell Signaling Technology, USA).

Cellular protein from HAECs or cells in frozen aorta tissue was prepared using RIPA buffer, followed by high-speed centrifugation and bicinchoninic acid (BCA) assay for protein quantification. Sodium dodecyl sulfate (SDS)-polyacrylamide gel (PAGE) electrophoresis followed by polyvinylidenedifluoride (PVDF) membrane transfer was employed for protein separation. The membranes were blocked with 5% non-fat milk containing PBS-Tween buffer blocking and incubated with primary monoclonal antibodies against SENP3 (Abcam, Cambridge, MA, USA), ICAM-1,VCAM-1 (Cell Signaling Technology, Cambridge, MA, USA), or internal loading control GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA, USA). After incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies, protein bands were developed with SuperSignal Ultra Chemiluminescent Substrate (Pierce, Rockford, IL, USA) using GE ImageQuant LAS4000mini system (GE, CA, USA).