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note
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2, 10 mM KCl, 0.1% NP-40, 0.5 mM DTT, 0.5 mM PMSF, 2 μg/ml pepstatin A, 2 μg/ml aprotinin and 2 μg/ml leupeptin). After the incubation on ice for 10 min, the samples were vortexed for 10 s and centrifuged at 5000 rpm for 1 min. Then the supernatant was collected as a cytoplasmic sample. Then the cytoplasmic protein (40 μg protein) was separated by 12% SDS-PAGE and blotted onto a PVDF membrane (GE Healthcare, Tokyo, Japan). The detection was performed using ECL Plus Western Blotting Detection System (ECL, GE Healthcare Bio-Science, Buckinghamshire, UK). Primary antibodies used were as follows: anti-Caspase3 (#9661S) (Cell Signaling Technology, Danvers, MA, USA) and anti-Actin (C-2)(Santa Cruz Biotechnology, Santa Cruz, CA, USA). Second antibody used was ECL anti-rabbit IgG Horse Radish Peroxidase linked whole antibody (GE Healthcare Bio-Science, Buckinghamshire, UK).
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