Fig. 2. JPH2 amino acids G482 and T483 are required for the formation of JPH2-CTP.

A) JPH2 cartoon showing full length protein with the antibody epitopes (top). Since JPH2-CTP is not detected by Ab1, the domain N-terminal of Ab1 is excluded (see 1). Based on western blotting, the molecular weight of JPH2-CTP is estimated to exceed 20kDa, hence the C-terminal domain is excluded (see 2). The domain spanning amino acids 465 to 510 (see 3) is predicted to contain the calpain cleavage site. Bioinformatic predictions of possible cleavage sites are shown, with the relative rankings below the sites marked by arrow heads. The site with the highest predicted score is colored red, the 2^nd-4^th highest prediction scores are colored blue. B) Western blots showing levels of full length JPH2 (JPH2-FL) and JPH2-CTP in lysates of HEK293 cells expressing wild-type JPH2, JPH2-CSD, and JPH2-ΔGT (in which residues ⁴⁸²G-⁴⁸³T were removed). All the experiments were performed at least 3 times to validate reproducibility.