Mast Cell Supernatants Induce Apoptosis

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).