Calpain and caspase-3 activity assay

Calpain activity was detected with a kit using Suc-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin (AMC) as a substrate (Calbiochem), as described previously [9]. AMC was measured at excitation and emission wavelengths of 380 nm and 460 nm, respectively. Calpain activity was quantified by the amount of free AMC per minute per microgram of protein. The activity of caspase-3 was measured with Biovision’s kit using N-Acetyl-Asp-Glu-Val-Asp-p-nitroanilide as a substrate. Free p-nitroanilide was quantified using a microtiter plate reader at 405 nm, and the data were normalized to the sham group, assigned a value of 1 [9].