CDK5/p25 kinase activity assay kit (Genmed, United States) was carried out according to the manufacturer’s protocols. The production of ADP was coupled to the oxidation of NADH using phosphoenolpyruvate (PEP) catalyzed by pyruvate kinase (PK) and lactic dehydrogenase (LDH). The cell lysate (100 μg) was incubated with the mixture including the PK/LDH system and the peptide substrate PKTPKKAKKL for 5 min at 30°C. The microplate reader (Molecular Devices, United States) was applied to examine the oxidation of NADH by measuring the absorbance at 340 nm. CDK5/p25 kinase activity was expressed as nmol NADH/min/mg of protein.
Calpain activity assay kit (Genmed, United States) was carried out according to the manufacturer’s protocols. The 200 μl mixture containing cell lysate (100 μg) was incubated for 60 min at 37°C with aminomethyl coumarin-labeled succinylated polypeptides, which is the substrate of calpain. Fluorescence produced by dissociative aminomethyl coumarin was detected by the fluorescence microplate reader (BioTek, United States) at excitation wavelength 380 nm and emission wavelength 460 nm.
Calpain activity assay kit (Genmed, United States) was carried out according to the manufacturer’s protocols. The 200 μl mixture containing cell lysate (100 μg) was incubated for 60 min at 37°C with aminomethyl coumarin-labeled succinylated polypeptides, which is the substrate of calpain. Fluorescence produced by dissociative aminomethyl coumarin was detected by the fluorescence microplate reader (BioTek, United States) at excitation wavelength 380 nm and emission wavelength 460 nm.