Figure 5
Calpain-specific cleavage fragments of JP2 are significantly increased in hiPSC-CMs after 2 months maturation, but not after 0.5 or 1 month differentiation. (A) Representative Western blot showing the protein levels of FL JP2 versus the Calpain-specific cleavage fragments. Both NT1 and CT1 were detected only after 2 months maturation, in contrast to the shorter 0.5 and 1 month hiPSC-CM differentiation time points. (B) Dot/bar plot quantifiying the Western blot data presented as mean ± SD (n = 4 biological replicates). Student’s t-test; *p < 0.05; ***p < 0.001. (C) Targeted data-dependent LC-MS/MS analysis of human Calpain-specific JP2 cleavage products in 2 months matured hiPSC-CMs. The relative abundance of the tryptic peptide peak areas of the endogenous human Calpain cleavage fragments is plotted versus the JP2 amino acid position including the primary human JP2 cleavage site R572 as indicated. (D) Sequence alignment confirming the high conservation of the primary Calpain cleavage site of JP2 across mammalian species from human to narwhal. Sequences human: NP_065166.2 (Homo sapiens); mouse: NP_001192005.1 (Mus musculus); rat: NP_001033063.1 (Rattus norvegicus); rabbit: NP_001075467.1 (Oryctolagus cuniculus); horse: XP_023482375.1 (Equus caballus); cat: XP_023106984.1 (Felis catus); goat: XP_017913386.1 (Capra hircus); Narwhal: XP_029077086.1 (Monodon monoceros). Asterisks mark identical residues; colons indicate highly conserved residues.
