Ab65308

The calpain and cathepsin D activity assay kits (ab65308 and ab65302, respectively) were both purchased from Abcam®. The activity assays were performed according to the manufacturer’s protocol using either an Envision® or Victor^{3 (link)} V Multilabel Plate Reader (both from Perkin Elmer). The 405 nm excitation and 515 nm emission filters were used in the calpain activity assay, and the 340 nm excitation and 460 nm emission filters were used in the cathepsin D activity assay. In each case the activity assays were performed in duplicate using cell lysates identical to those used in SILAC-SPROX analyses. In the calpain activity assay, fluorescence measurements were recorded on ~30 µg of the total lysate in the absence and presence of 0.5 µl of the calpain inhibitor provided with the kit. In the Cathepsin D activity assay, fluorescence measurements were recorded on ~20 µg of the total lysate in the absence and presence of 10 µl of a 1mM stock solution of pepstatin A. In each case the specific activity of the enzyme was measured by subtracting the fluorescence intensity of the sample with the inhibitor from the one without the inhibitor and normalizing to the total protein concentration used in each reaction.

Calpain activity was determined in platelet lysates incubated in 5mM glucose DMEM for 6 hours. After 6 hours, platelets were centrifuged at 13,000g × 5 minutes then lysed in assay buffer and analyzed for calpain activity (ab65308, Abcam, Cambridge MA), which was normalized to protein content. In vitro, calpeptin was co-incubated with platelets incubated in 5mM glucose DMEM for 6 hours and analyzed for annexin V binding. In vivo, calpeptin was administered I.P. at 7mg/kg, in 5% DMSO: Saline (100μL final volume) for 7 days. Mice were then pulsed with Anti-GP1bβ-FITC antibody and calpeptin administration continued. Blood was analyzed daily.