Nbp1 80306

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).

For detecting C-Myb, BCL-2, CD31, or CD34 protein level in artery walls, IHC was performed in human or rat paraffin-embedded 5-µm-thin sections as previously described^{13 (link), 14 (link))}. Sections were incubated with primary antibodies for C-Myb (NBP1-80306, Novus, USA), BCL-2 (NB100-92142, Novus, USA), CD31 (ab28364, Abcam, UK), and CD34 (ab81289, Abcam, UK). After treatment with EnVision™ Detection Systems Kit, Peroxidase/DAB, Rabbit/Mouse, slides were visualized and photographed using Olympus BX53 microscope (Olympus, Japan) and analyzed using Image-Pro Plus 6.0 software (Media Cybernetics, Inc.).