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Treatment of SK-N-BE(2) lysate with caspases
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SK-N-BE(2) cells (1 × 106 cells for each sample) were lysed by repeated passage through a 26-G syringe in the reaction solution recommended by the manufacturer (50 mM HEPES pH 7.2, 50 mM NaCl, 0.1% CHAPS, 10 mM EDTA, 5% glycerol and 10 mM DTT) and then separately incubated with 2 U of active human caspase-3, -6, -7 or -8 for 180 min at 37 °C. The cleavage reaction was terminated by adding a buffer constituted by 50 mM Tris pH 6.8, 5% (w/v) SDS, 8 M deionized urea, and 2% (v/v) 2-mercaptoethanol. Samples were then frozen at − 80 °C.
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