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To map the calpain cleavage site, we implemented two approaches.
To precisely map the calpain cleavage site, we performed in vitro calpain cleavage reactions using purified recombinant HIS6-SMN/GST-Gemin2 heterodimers followed by mass spectrometric analysis of the C-terminal cleavage product.
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Protein 2.2 is distinct from the calpain fragment based on peptide maps and reaction with domain-specific antibodies.
H.M. and S.H. carried out the calpain and ubiquitination analyses.
Immunofluorescence analyses revealed that calpain-1 localizes to filopodia (Fig. 7d) and ratiometric FRET analyses of cells co-expressing a calpain sensor33 and MYO10-mCherry demonstrated that calpains are active within filopodia (Fig. 7e; controls related to the use of the calpain FRET probe are shown in Supplementary Fig. 11).
Calpain inhibitors inhibit the calpain activity, resulting in reducing the calpain-meditated apoptosis[10].
The calpain inhibitor calpeptin prevents MV-induced muscle atrophy.
In contrast, animals pretreated with the calpain inhibitor showed minimal evidence of apoptosis.
Our data suggest that a complimentary cross talk exists between the calpain system and the UPP.
The calpain inhibitor SNJ-1945 was kindly provided by Senju Pharmaceutical Co., Ltd.
The calpain activity and calpain-II (m-calpain) protein (but not calpain-I (µ-calpain)) level could also be increased by honokiol.
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