In vitro and cell-based calpain cleavage assay

For in vitro calpain cleavage assays, the vector encoding T7-tagged mouse cdr2 in pcDNA3 was transcribed and translated in the presence of [³⁵S]-methionine (Perkin Elmer, Boston, MA, USA) using a TnT Quick coupled transcription/translation system (Promega, Madison, WI, USA) according to the manufacturer's recommendations. For cell-based cleavage assay, MN9D cells were lysed in buffer containing 50 mM Tris-HCl, pH 8.0, 2 mM EDTA, and 1% Triton X-100 buffer without protease inhibitor cocktail. [³⁵S]-cdr2 or cell lysates (50 μg) were incubated for 1 h at 30 °C in a calpain activation buffer containing 1 mM CaCl₂ in the presence or absence of purified m-calpain (0.343 units) or μ-calpain (0.134 units; both from Calbiochem) as recommended by the manufacturer. If necessary, calpeptin (50 μM) or MG132 (2.5 μM) was added to the reaction mixtures. Reactions were terminated by the addition of 5 × protein sample buffer followed by boiling for 5 min. The resulting products were separated on 10% SDS-PAGE gel and processed for autoradiography or immunoblot analysis.