Figure 4

Casp6WT and Casp6N73T enzymatic processing of Lamin A/C. (a) Western blot of full-length (#2032 Cell Signaling Technology; top) or cleaved Lamin A/C at VEID (neoepitope antibody #2035 Cell Signaling Technology; middle) and β-Actin (bottom) from 50–500 nM recombinant Casp6WT or Casp6N73T incubated with Casp6 knockout mouse tissue nuclear extracts. (b) Quantification of full-length Lamin A/C and cleaved Lamin A/C by Casp6WT or Casp6N73T expressed as % of the control with no added recombinant active Casp6. (c) Western blots of time-dependent cleaved Lamin A/C by 200, 300, 400, or 500 nM Casp6WT or Casp6N73T incubated with Casp6 knockout tissue extracts. (d) Quantification of time-dependent generated cleaved Lamin A/C by Casp6WT or Casp6N73T shown in (c) expressed as % of the control with no added recombinant active Casp6. Insets: The initial velocity phase of the cleavage curve. The linear regression was performed on % cleaved Lamin A/C at 5, 10, and 15 min by 200 nM Casp6 (WT: R² = 0.82, N73T: R² = 0.82), or on % cleaved Lamin A/C at 5 and 10 min by Casp6 with 300 nM (WT: R² = 0.88, N73T: R² = 0.80), 400 nM (WT: R² = 0.71, N73T: R² = 0.66), or 500 nM (WT: R² = 0.90, N73T: R² = 0.90). (e) The initial velocity of Casp6WT and Casp6N73T reacting on Lamin A/C measured from (d). Data were shown as mean ± s.e.m from 3 independent experiments. Statistical evaluations were done with two-way ANOVA (variant: p = 0.0001, concentration: p < 0.0001, interaction: p = 0.1329). Post-hoc analyses were done with Tukey’s test. ***p < 0.001 N73T vs WT. Full-length images of blots/gels are presented in Supplementary Information.