Fig 1: Caspase-1 activation during Eh-macrophage contact degrades cullin-1/5.(A, B) THP-1 derived macrophages were pre-incubated with the pan-caspase inhibitor Z-VAD-fmk (100μM) and caspase-1 specific inhibitor Z-YVAD-fmk (100μM) for 1 h followed by stimulation with Eh (10:1 ratio) for 10 min. Cleavage of the cullin-1/5 were assessed by western blot. (C, D) Wild type (WT) THP-1 and CASP1 CRISPR/Cas9-KO macrophages were stimulated with Eh (10:1 ratio) for 10 min. (E, F) WT, THP-1 def ASC and NLRP3 CRISPR/Cas9-KO THP-1 macrophages were stimulated with Eh (10:1 ratio) for 10 min. After incubation, cells were washed and lysed. Equal amount of protein was loaded on to the SDS-PAGE (7.5%) and immunoblotted with anti-cullin-1 (Panel: A, C, and E), anti-cullin-5 (Panel: B, D and F) and anti-GAPDH antibody. (G) Cullin-1 was immunoprecipitated (Cul-1 IP) using anti cullin-1 antibody and post immunoprecipitation it was incubated with recombinant caspase-1 (C-1) for 4h or overnight (O/N). (H) Immunoprecipitated cullin-1 was incubated with recombinant caspase-1 or with Y-VAD-fmk (50μM or 100μM) or Z-VAD-fmk (100μM). (I) Immunoprecipitated cullin-5 was incubated with recombinant caspase-1 or with Z-VAD-fmk (100μM) or Y-VAD-fmk (100μM). (J) Recombinant cullin-1 (rec Cul-1) was incubated with recombinant caspase-1 overnight at 37° and was immunoblotted with anti-cullin-1 antibody. Note, recombinant cullin-1 (rec Cul-1) has a molecular weight of ~118 kDa due to the N-terminus GST tag (~28kDa). The highlighted boxed areas in the figures show point of interest for cullin-1/5 as described in text. (K) Protein-protein interaction using STRING v11 showed direct interaction between cullin-1, cullin-5 and Nedd8 demonstrated that cullin-1/5 are novel substrates for caspase-1. Data (A, B, C and D) are representative of three independent experiments while data (E, F, G, I, and J) are representative of two independent experiments.