FIGURE 6.

MPP⁺-mediated cleavage of the identified substrates is attenuated by calpeptin. A, MN9D cells were incubated with or without 50 μm MPP⁺ for 36 h. Cells were stained with 3 μm Fluo-3/AM dye and examined under a fluorescence microscope equipped with epifluorescence filters and a digital analyzer. Scale bars represent 50 μm. B, to measure calpain activity in MN9D cells treated with 50 μm MPP⁺ in the presence or absence of 50 μm calpeptin for the indicated time periods, cellular lysates (50 μg) were separated and processed for immunoblot analysis using an anti-fodrin antibody. C–E, MN9D cells were incubated in 50 μm MPP⁺ in the presence or absence of 50 μm calpeptin for the indicated time periods. Cellular lysates (100 μg) were subjected to immunoblot analysis using the indicated antibodies. Changes in fold intensity were calculated as described in the legend Fig. 5. All values represent the means ± S.E. from three independent experiments. ***, p < 0.001; **, p < 0.01. ns, not significant; Cont., control.