Figure 5

Casp6WT and Casp6N73T enzymatic processing of α-Tubulin. (a) Western blot of full-length (11H10 Cell Signaling Technology; top) or cleaved α-Tubulin at VGVD438 (GN20622; bottom) in the reactions of 15.6–250 nM recombinant Casp6WT or Casp6N73T with 242 nM of purified α- and β-Tubulin (121 nM of α-Tubulin). Tub∆Casp6, α-Tubulin cleaved by Casp6. (b) Quantification of Tub∆Casp6 shown in (A) expressed as % of FL Tubulin. Statistical evaluations were done with two-way ANOVA (variant: p = 0.0002, concentration: p < 0.0001, interaction: p = 0.4555). Post-hoc analyses were done with Tukey’s test. ***p < 0.001 N73T vs WT. (c) Western blot of time-dependent cleaved 121 nM α-Tubulin by 15.6, 31.2, or 62.5 nM Casp6WT or Casp6N73T. (d) Quantification of Tub∆Casp6 shown in (c) expressed as % of FL Tubulin. Insets: The initial velocity phase of the cleavage curve. The linear regression was performed on % Tub∆Casp6 at 1, 2, 3, and 4 h by 15.6 nM Casp6 (WT: R² = 1.00, N73T: R² = 0.99), on % Tub∆Casp6 at 1, 2, and 3 h by Casp6WT with 31.2 nM (R² = 0.99) or 62.5 nM Casp6WT (R² = 1.00), or on % Tub∆Casp6 at 1 and 2 h by Casp6N73T with 31.2 nM (R² = 0.99) or 62.5 nM (R² = 1.00). (e) Initial velocity of Casp6WT and Casp6N73T reacting on α-Tubulin measured from (d). Statistical evaluations were done with two-way ANOVA (variant: p < 0.0001, concentration: p < 0.0001, interaction: p = 0.0015). Post-hoc analyses were done with Tukey’s test. ****p < 0.0001 N73T vs WT. Data represents mean ± s.e.m. from 3 independent experiments. Full-length images of blots/gels are presented in Supplementary Information.