GoKawiila, CASP5 and FLAG immunoblot of HEK293T expressing various CASP5 constructs as indicated with or without GSDMD at 24 hr post transfection. b, CASP5 activity in HEK293T cell culture medium expressing indicated CASP5 isoforms or 2-fold (1/2) or 4-fold (1/4) less CASP5c and co-expressing GSDMD. c, TOPflash luciferase reporter activity (left panel) and corresponding immunoblots (right panels) in HEK293T cells expressing indicated CASP5 isoforms or 2-fold (1/2) or 4-fold (1/4) less CASP5c. d, CASP5 activity in culture medium of HEK293T cells treated with or without 20 μM WEHD, LEVD or Q-VD-OPh inhibitor for 18 hr. e,f,h,j, Immunoblots for CASP5 in WCE from CASP5a, CASP5b, CASP5c, or CASP5c (C173A) expressing HEK293T cells (e), or CASP5 isoform expressing HEK293T cells without (Ctrl) or treated with 20 μM WEHD, LEVD,VX765 or Q-VD-OPh inhibitor for 18 hr (f,h,j). Vector indicates an empty vector without the insertion of the CASP5 gene (f,h,j). Autocleavage products manifested in the appearance of N-terminal fragments CASP5a-N (38 kDa), CASP5b-N (31 kDa), and a smaller CASP5c-N (22 kDa), depended on catalytic activity, and were absent upon transfection of mutant CASP5c (C173A). The sizes of the autocleavage products were concordant with predicted proteolytic cleavage sites4. g,i,k, Schematics of predicted CASP5a, CASP5b and CASP5c auto-cleavage steps and cleavage fragments. CASP5a, b, and c share a common domain structure composed of caspase protease domain which is composed of large (p20) and small (p10) subunits separated by an interdomain linker (IDL). The N-terminal CARD (blue) in CASP5a and CASP5b is connected to the protease domain via a CARD domain linker (CDL). l, Immunoblots for CASP5 in WCE from CASP5c or CASP5-ΔCARD expressing HEK293T cells. m, Amino acid sequences of CASP5a and CASP5c highlighting the location of the C residue that were mutated to A in this study: C315A in CASP5a and the equivalent C173A in CASP5c. n, CASP5 substrate prediction among Wnt signaling pathway proteins and regulators using the PeptideCutter software (https://web.expasy.org/peptide_cutter/), caspase 1 was employed as the probe in the prediction. o, Immunoblotting analysis of truncated APC cleavage by CASP5c in HEK293T cells. p, Schematics showing the truncated APC relative to full length APC and predicted fragments post cleavage at D566. q, Quantification of Fig. 3n AXIN IP indicated immunoblot relative band intensities from WNT3A-CM treated (2 hr) APC-KO HEK293T cells with ectopic expression of APC-FL or APC-D556A and CASP5c as indicated, Band intensities were calculated with ImageJ. r, TOPflash luciferase reporter activity from APC-KO HEK293T cells with ectopic expression as indicated, harvested 48 hr post-transfection after overnight WNT3A-CM treatment. s, Quantification of Fig. 3o AXIN IP indicated immunoblot relative band intensities from APC-KO HEK293T cells without (Ctrl) or with ectopic expression of APC-FL, APC-1-556, or APC-557-2843. Band intensities were calculated with ImageJ. t, TOPflash luciferase reporter activity from WT or APC-KO HEK293T cells expressing indicated plasmids for 48 hr. a,c,e,f,h,j,l,o, ACTB is β-actin loading control. All CASP5 encoding plasmids transfected at 100 ng/24 well plate and WCE or supernatants prepared at 24 hr post-transfection. b, n = 6, c,d,r–t, n = 3, q,n = 2-3. One-way ANOVA with Tukey’s post-hoc test (b–d,q–t); Two-tailed unpaired t-test (q); Error bars, mean ± s.e.m.
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