Identification of a peptide fragment of DSCR1 that competitively inhibits calcineurin activity in vitro and in vivo

Chan et al. 10.1073/pnas.0503846102.

Supporting Information

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Supporting Methods
Supporting Figure 6
Supporting Figure 7
Supporting Figure 8

Supporting Figure 6

Fig. 6. Architecture of DSCR1. Multiple sequence alignment of human DSCR1 and its orthologues generated by using the clustal algorithm. Exons 5, 6, and 7 (arrows) are shared by all calcipressin isoforms. The signature motif of the calcipressins, FLISPPXSPP, is encoded by exon 6 and is shown in a dotted box. Another conserved motif, PXIIXT, encoded by exon 7, is indicated by the solid box. (human: Homo sapiens, sheep: Ovis aries, cow: Bos taurus, dog: Canis familiaris, mouse: Mus musculus, rat: Rattus norvegicus, frog: Xenopus laevis, zebrafish: Danio rerio, honey bee: Apis mellifera, fly: Drosophila melanogaster, worm: Caenorhabditis elegans, yeast: Saccharomyces cerevisiae).

Supporting Figure 7

Fig. 7. CD spectra of DSCR1-ex7. (A) UV CD spectra of a 0.2 mg/ml solution of DSCR1-ex7 in 10 mM sodium phosphate pH 7.5, 50 mM NaSO₄, and 2 mM DTT indicate that a large fraction of the protein adopts a random-coil conformation. (B) Thermal melting curves of a 0.2 mg/ml solution of DSCR1-ex7 in 10 nM Na phosphate pH 7.5, 50 mM NaSO₄, and 2 mM DTT were obtained by using the following acquisition parameters: temperature range, 10-95°C; equilibration time, 30 s; averaging time, 15 s; data interval, 1°C; spectral bandwidth, 1.5 nm; scan range, 222 (●) and 205 (○) nm.

Supporting Figure 8

Fig. 8. Kinetic analysis of the inhibitory mechanism of DSCR1 using full-length calcineurin. (A) Activity of calcineurin toward pNPP was measured. The K_m value for pNPP ranged from 50 mM to 216 mM in different preparations of purified CNa . (B and C) CNa was incubated with full-length DSCR1 (B) and DSCR1-ex7 fragment (C) at different concentrations [0 nM (filled circles), 10 nM (empty circles), 25 nM (filled triangles), and 100 nM (open triangles) in pNPP concentrations increasing from 10 to 150 mM. Each graph represents the means ± SD from triplicate reactions. The K_i values were calculated graphically from the reciprocal plots. K_i values for DSCR1 and DSCR1-ex7 range from 7.6–84.6 nM and 12.8–89.3 nM, respectively.