FIGURE 4.

Cell-based calpain cleavage assays. Lysates from MN9D cells overexpressing T7-ASNA1-FLAG (A), OPTN-GST (B), or T7-PERI-FLAG (C) were incubated for 2 h at 37 °C with the indicated calpains (5.55 unit m-calpain or 3.95 units μ-calpain) in the presence or absence of 500 μm PD150606. After separation by SDS-PAGE, gels were processed for immunoblot (IB) analysis using anti-T7, -GST, or -FLAG antibody, respectively. Closed and open arrowheads indicate full-length and fragments of each protein, respectively. Fodrin and GAPDH were used as the calpain activation and loading control, respectively. To examine whether cleavage of ASNA1 (D), OPTN (E), and PERI (F) was achieved in a calpain concentration-dependent manner, all protein lysates (100 μg) from MN9D cells transiently overexpressing tagged proteins were incubated with increasing doses of m-calpain (1.11–5.55 units) in the presence or absence of 500 μm PD150606 or 50 μm calpeptin. Immunoblot analyses were performed using anti-T7, -GST, or -FLAG antibody. The asterisk in F indicates a nonspecific band. Representative blots from three independent experiments are shown.