Figure 4.

Effect of calpain concentration, calcium, and calpain inhibitor on calpain activity in vitro: (A) Calpain 1 (C1) or 2 (C2) were incubated with P1 polyprotein at +25 °C for 2 h. The reactions included 2 mM CaCl₂ and calpains with increasing catalytic activities: 0.01, 0.1, 0.5, or 1 unit. Also, samples without any proteases or CaCl₂ or with 4 mM EGTA were included. (B) Titration of CaCl₂ against 1 unit of calpain 1 or 2. The reactions were carried out as in Figure 4A. (C) The effect of calpain inhibitor (ci) or elastatinal (ela) on calpain activity during P1 processing: P1 was treated with 1 unit of calpain 1 (C1) or 2 (C2) in the presence or absence of 200 µM calpain inhibitor (ci) or 250 µM elastatinal (ela) for 2 h at +25 °C. Representative images of P1, VP1, and VP3 bands. (D) Quantification of the VP1 and VP3 band intensities from Figure 4C: Data are presented as means ± SEM from three separate experiments. Significance was determined with one-way ANOVA with Bonferroni’s multiple comparisons test (* p < 0.05; **** p < 0.0001; ns, nonsignificant).