Caspase 9 h 170

aHSCs were treated as indicated, detached, thoroughly washed with PBS, and then lysed in ice-cold lysis buffer. Following centrifugation at 13,000 ×g for 10 min at 4°C, the supernatants (30 μg protein) were boiled with reducing sample buffer for 5 min, subjected to electrophoresis in SDS-polyacrylamide gels, and then transferred onto a PVDF membrane. The membrane was blocked with 1% BSA in PBS containing 0.1% Tween-20 (PBST) for 1 h at room temperature and then washed with PBST. Proteins were detected by incubating the membrane overnight at 4°C with antibodies against α-SMA, β-actin (Sigma-Aldrich, MO), Bak, Bal-2 (C21), Bax, caspase-3 (H-277), caspase-9 (H-170) (Santa Cruz Biotechnology, CA), cleaved caspase-9 (Asp 353), and cleaved PARP (Asp 214) (Cell Signaling Technology, MA). Next, the membrane was incubated with a primary antibody, and, finally, the membrane was incubated with a secondary antibody conjugated to horseradish peroxidase (HRP) for 1 h. An enhanced chemiluminescence (ECL) kit (Amersham Biosciences, IL, or Millipore, MA) was used for protein detection. The relative intensity of the immunoreactive bands was assessed using Image J software.

Materials purchased from Sigma Chemical Co. (St. Louis, MO, USA) were thymol, DMSO (dimethyl sulfoxide), DCFH-DA (2′,7′- dichlorofluorescein-diacetate), AO/EB (acridine orange/ethidium bromide) stain, SDS polyacrylamide gels, coomassie brilliant-blue-dye, powdered skim milk, trypan blue, comet assays, low-melting agarose, normal melting agarose, and lysis solution. Ham’s F-12 culture medium, fetal bovine serum (FBS), and antibiotics (100 U/mL penicillin, 100 μg/mL streptomycin) were obtained from Gibco Invitrogen Corporation (Carlsbad, CA, USA). CellTiter-Glo Luminescent Cell Viability and GSH/GSSG-Glo assays were provided by Promega (Madison, MI, USA). Bax (N-20), Bcl-2 (C-21), Caspase-3 (H-277), Caspase-9 (H-170), and β-actin (AC-15) antibodies were purchased from Santa Cruz Biotechnologies (Santa Cruz, CA, USA). Amersham ECL Plus Western Blotting Detection reagents were obtained from GE Healthcare (Piscataway, NJ, USA). Radioimmunoprecipitation assay (RIPA) buffer and a proteinase inhibitor cocktail were from Roche (Mannheim, Germany). Molecular weight marker BenchMark Pre-Stained Protein Ladder was from Invitrogen (Grand Island, New York, USA).