Overview of CAPN3 substrates and tested peptides.

Upper part: List of described CAPN3 substrates in which the putative CAPN3 cleavage motif was identified. For each substrate the peptide motif is shown, with key residues spaced and underlined. The peptides were tested for cleavage potential in a BG fusion protein and judged as cleaved (+), partially cleaved (+/−), or not cleaved (−). In Figure 1F BGn (with peptide from AHNAK-N) is a good (+) substrate, BGc1 and -c2 are moderate (+/−), and BGr is bad (−). In addition, the protein and gene ID are given, the location of the motif, and the literature reference. Titin952–1540 and AHNAK-R were described as non-substrates and contain no motif sequence. For AHNAK-R a peptide with Blast homology to the peptide from AHNAK-N is shown, with in bold the residue that is not conform the cleavage motif. Middle part: Single mutants of the AHNAK-N and FLNC peptides were analyzed for cleavage potential. Mutated residues are shown in bold. Lower part: Several candidate peptides conforming to the general motif or the specific motif were analyzed for cleavage potential. Most peptides were also tested in a reverse orientation of the fusion proteins (GB, N-terminal GFP and C-terminal β-Galactosidase), never to give a discrepant result. Representative western blots for table 1 are included in Figure S1.