Figure 6

Steady levels of Casp6N73T and Casp6WT proteins in transfected HEK293T cells. (a) RedSafe-stained agarose gel of human Casp6 and Hprt1 mRNA amplicons in non-transfected (NT), mock, pCEP4β, pCEP4β-CASP6C163A-His, pCEP4β-CASP6WT∆Pro-His, or pCEP4β-CASP6N73T∆Pro-His-transfected cells. (b) Quantification of Casp6 mRNA levels normalized to Hprt1 shown in (a). (c,e) Western blot and (d,f) quantification of Casp6 in transfected cells against Pharmingen (c,d) or 10630 neoepitope antibodies (e,f). ReWT recombinant Casp6WT, ReC163A recombinant Casp6C163A, FL full length, ∆Pro Casp6 without pro-domain, SS small subunit, LS large subunit. Statistical evaluations were done with one-way ANOVA (d,f: p < 0.0001) followed by post-hoc Tukey’s test (**p < 0.01, ****p < 0.0001). (g) VEIDase activity in protein extract from transfected cells. Statistical evaluations were done with one-way ANOVA (p < 0.0001) followed by post-hoc Tukey’s test. ****p < 0.0001 WT vs NT. (h) Western blot and (i) quantification of Casp6 in transfected cells with the Pharmingen antibody. The levels of ∆ProCasp6 normalized to the 0 h were fit to a one-phase decay to calculate the decay rate of Casp6WT or Casp6N73T. * indicates p = 0.012 done with unpaired t-test. Data shown represents mean ± s.e.m. from 3 independent experiments. Full-length images of blots/gels are presented in Supplementary Information.