Anti mouse bcl 2 antibody

Cells were pretreated with activated or resting mast cell supernatants for 24 h. Cell lysate preparation was carried out as described (35 (link)). In brief, cells were lyzed using the lysis solution (50 mM Tris, 1% Triton X-100, 0.1% SDS, 150 mM NaCl) containing protease and phosphatase inhibitors (5 mM iodoacetamine, 50 mM PMSF, and 0.1 mM TLCK) (Thermo Scientific). The cell lysate was centrifuged at 20,854 g for 30 min. Protein were quantified using Bradford reagent and 40–60 μg protein was resolved on 12% SDS PAGE and transferred onto the polyvinylidene difluoride (PVDF) membrane. Blocking of membrane was carried out for 1 h by tris-buffered saline containing 0.05% Tween-20, and 5% (w/v) non-fat dry milk. Overnight incubation with primary antibody was carried out at 4°C. Primary antibodies used are anti-mouse PARP antibody (Cell Signaling Technology, Danvers, MA), anti-mouse Survivin antibody (Cell Signaling Technology, Danvers, MA), anti- mouse Caspase-3 antibody (Cell Signaling Technology, Danvers, MA), anti- mouse BCL-2 antibody (Cell Signaling Technology, Danvers, MA). After which incubation with secondary antibody was carried out, the immune-reactive bands were visualized using enhanced chemiluminescence method. The blots were then washed and re-probed with loading control, anti-β-actin antibody (Sigma).

Acetylshikonin (CAS Number: 24502-78-1) and simvastatin (CAS Number: 79902-63-9) were purchased from Chem Face China Company. Temozolomide (CAS Number: 85622-93-1), propidium iodide (CAS Number: 25535-16-4), 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) (CAS Number: 298-93-1), acridine orange (CAS Number: 65-61-2), bafilomycin A1 (Cat#B1793-10UG) and anti-rabbit LC3β antibody (Cat#L7543—100UL) were purchased from Sigma-Aldrich Co. Anti-rabbit p62 antibody (Cat#39749), anti-rabbit Beclin-1 antibody (Cat#3738), anti-mouse Bcl2 antibody (Cat#15071), anti-rabbit Mcl-1 antibody (Cat#4572), anti-rabbit Bcl-XL antibody (Cat#2762), and anti-rabbit GAPDH antibody (Cat#2118) were purchased from Cell Signaling Company. The secondary antibodies, anti-rabbit HRP-conjugate and anti-mouse HRP-conjugate, were purchased from Sigma-Aldrich (Oakville, ON, Canada) as well. The enhanced chemiluminescence (ECL) (CAS Number: 12630) was acquired from Cell Signaling Technology Co. (Beverly, MA, USA). The bicinchoninic acid (BCA) protein assay kit was obtained from Thermo Fisher Scientific (Winnipeg, MB, Canada).

The fixed hearts and abdominal aortas were embedded in paraffin and sectioned. Step sections in the horizontal direction were made every 10 μm, to observe the histological changes in the coronary artery, aortic root, and abdominal aorta in detail. Haematoxylin and eosin (H&E) staining was performed to observe the inflammatory changes, by means of light microscopy. Verhoeff's van Gieson (EVG) staining was used to investigate the vascular architecture. Immunohistochemical analysis was performed to detect the expression level of Bcl‐2 using an anti‐mouse Bcl‐2 antibody (1:200, Cell Signalling Technology). The stained sections were photographed under a light microscope (Leica).