Fig. 4. Functional switch of cathD activities relies on pH and its maturation.

Representative immunoblot images (a) and quantification (b) of cathD-mediated cofilin phosphorylation in different pH conditions, showing both recombinant cathD^wt and cathD^D231N (10 nM) dephosphorylate cofilin in neutral pH (7.5), whereas cathD^wt mainly degrades cofilin in acidic pH (3.5). Note that both cathD^wt or cathD^D231N significantly reduce p-cofilin/cofilin ratio in neutral but not acidic pH (c). Representative immunoblot images (c) and quantification (d) of cathD-mediated cofilin phosphorylation in flies. c Elevated p-cofilin levels in cathD¹ mutants are restored upon expressing either wild-type (cathD¹; da > cathD^wt) or proteolytically inactive cathD (cathD¹; da > cathD^D231N). d Quantification of p-cofilin/total cofilin levels (normalized to w¹¹¹⁸ controls). The ratio is reduced when cathD^wt or cathD^D231N is expressed in cathD¹ mutant background. Representative immunoblot images (e) and quantification (f) of in vitro phosphatase assay showing that immature recombinant human cathD directly dephosphorylates cofilin at pH 7.5, whereas mature cathD degrades cofilin at pH 3.5 (e). f Quantifications of p-cofilin/cofilin ratio (normalized to control) show that immature cathD dephosphorylates cofilin, whereas mature cathD shows limited phosphatase activity in neutral pH (7.5). Representative immunoblot images (g), quantification of p-cofilin/cofilin ratio (normalized to cathD^wt, h) and cofilin level (normalized to cathD^wt, i) from in vitro phosphatase assays using recombinant cathD^wt, cathD^D231N, cathD^D33G, cathD^D75G, and cathD^E117. Note that cathD^D33G, cathD^D75G and cathD^E117G exhibit impaired activities in cofilin dephosphorylation at pH 7.5, whereas cathD^D33G and cathD^D231N present suppressed proteolytic activities for cofilin degradation at pH 3.5. Data are means ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001 by Mann–Whitney U test or One-way ANOVA with Tukey’s post hoc test.