Polyclonal rabbit antibodies to caspase-3 (Santa Cruz
Biotechnology, Santa Cruz, CA) were used. Cells were washed with
ice-cold PBS and lysed in buffer containing 20 mM Tris-HCl
(pH7.4), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM
sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM
Na3VO4, 1 μg/ml
leupeptin, and 1 mM phenylmethylsulfonyl fluoride. Protein levels were
measured by a protein assay kit (Bio-Rad, Richmond, CA). Proteins were
separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis
(SDS-PAGE; 15% PAGE) and transferred onto polyvinylidene difluoride
(PVDF) membrane (Schleicher & Schuell, Dassel, Germany). Membrane was
blocked with 5% nonfat dry milk for 1 hour and incubated with primary
antibody for overnight. The secondary antibody was goat anti-rabbit IgG
(Amersham–Pharmacia Biotech, Uppsala, Sweden) conjugated to
horseradish peroxidase. Enhanced chemiluminescence (ECL;
Amersham-Pharmacia Biotech) was used for the detection of protein
signals. Enhanced luminescence of luminol by peroxidase-catalyzed
oxidation was detected by autoradiography (Hyperfilm ECL;
Amersham-Pharmacia Biotech).