Fig. 1

OMP was unlikely to be cleaved by CAPN5.

a, Primary structure of mouse OMP. The asterisks indicate the highly scored residues in three prediction models: Model 1, support vector machine (SVM) with radial basis function kernel; Model 2, SVM with linear kernel; Model 3, protein secondary structure prediction. See Methods for details. All models predict a possible cleavage at K33 (arrow). b, Plasmids and molecular diagrams for Myc-OMP-His and CAPN5-His with approximate molecular weights in kDa. N and C; N- and C-termini; ires, internal ribosomal entry site. c-e, Confirmation of concomitant immunoreactivity (IR) to the fusion myc-OMP-His protein expressed in HEK293T cells: (c) OMP-IR and Myc-IR; (d) Myc and His-IR; (e) OMP-IR and His-IR. f,g. CAPN5 and His expression was confirmed by GFP coexpression because the antibodies were both raised in mice: (f) CAPN5-IR and GFP fluorescence and (g) His-IR and GFP fluorescence. Scales, 20 μm in (c-g). DAPI, 4’,6-diamidino-2-phenylindole. h, i, Confirmation of purification of His-tagged purified (h) Myc-OMP-His (22 kDa) and (i) CAPN5-His (74 kDa) by western blotting. j-l, Size of Myc-OMP-His by (j) Myc-IR, (k) OMP-IR and (l) His-IR after incubation with CAPN5-His in the presence of different concentrations of Ca²⁺. No apparent fragmentation of OMP was observed below 22 kDa. Note that cleavage at K33 in OMP should result in two fragments: 5 kDa (Myc-tagged N-terminus) and 16 kDa (His-tagged C-terminus). m. Summary of the western blot intensities for Myc-, OMP- and His-IRs (n = 4). No significant changes in these intensities were detected. n, o, Western blot of the cell lysate of HEK293T cells expressing (n) Myc-OMP-His and (o) CAPN5-His in the absence (Ctrl) and presence of Ca²⁺-ionophore (1 µM; Iono) for 12 h. p, q, Summary of western blot intensities for (p) Myc-IR and (q) His-IR (n = 3). No smaller fragments were observed below 22 kDa, corresponding to the uncut Myc-OMP-His. Mean ± SD.

STATISTICS: (m) One-way ANOVA with a post hoc Tukey‒Kramer comparison among different Ca²⁺-concentration groups (0–3 mM): Myc; F(4,19) = 1.185, P = 0.35696: His; F(4,19) = 0.884, P = 0.497: OMP; F(4,19) = 0.977935, P = 0.44871. No significant difference was detected (NS). The actual values from the post hoc test are shown in the Supplementary Data. (p,q) Paired, two-tailed Student’s T test for the comparison between Ctrl and Iono; P = 0.821 for Myc, P = 0.6 for His.