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. 2021 Jan 5;11:1. doi: 10.1038/s41398-020-01158-w

Fig. 5. The effect of PIDD1 mutations on interacton with CRADD and caspase-2 activation.

Fig. 5

A PIDD1 immunoprecipitation pull down of CRADD. HEK293T cells transfected with vectors encoding GFP or FLAG-tagged WT or mutant PIDD1 were lysed after 24 h, and subjected to immunoprecipitation, followed by western analysis using antibodies recognizing the FLAG-tagged PIDD1, or CRADD, in the IP sample or flow through. B Functional effect of PIDD1 mutation on Caspase-2 activity through MDM2 cleavage assay. PIDD1-deficient U2OS or A549 cells generated by CRISPR/Cas9 technology were transiently transfected with the same constructs used in A and treated with the AuroraB kinase inhibitor ZM447439 to induce cytokinesis failure and PIDDosome activation. Cell lysates were probed with an antibody recognizing the cleaved variant of MDM2. Reprobing with an antibody recognizing GAPDH was done to confirm comparable protein loading.