Calpain activity assay.

Calpain activity was assessed using a commercially available kit (Abcam, Cambridge, MA), as previously described (27, 37). This kit includes a proprietary set of buffers to allow determination of calpain activity in tissues before homogenization. In brief, samples were prepared in the proprietary extraction buffer according to the manufacturer's specifications. Sample protein levels were measured, and 100 μg of protein from each sample were diluted to a final volume of 85 μl with extraction buffer. An additional aliquot of each sample (100 μg of protein) was mixed with 1 μl of calpain inhibitor and sample volume brought to 85 μl with extraction buffer. Extraction buffer alone was used as a blank. All mixtures were placed in a 96-well plate, and then reaction buffer (10 μl) and calpain substrate (5 μl) were added to wells. An initial reading was taken, plates were incubated in the dark at 37°C for 1 h, and a final fluorescent reading was made (excitation 400 nm, emission 505 nm). The difference in increases in fluorescent activity over time for a given sample in the presence and absence of the specific calpain inhibitor was taken as an index of calpain enzyme activity.