DFNA5 is dispensable for secondary necrosis after NLRC4/caspase-8-mediated cell death. (a) Immunoblot of mouse DFNA5 and actin from iMac whole cell lysates. (b–e) Casp1−/−Casp11−/− iMac expressing Dfna5, Asc or luciferase (control) sgRNA were subjected to electroporation with flagellin (0.5 μg ml−1) and cytochrome-c (50 μg ml−1), or treated with FasL (100 ng ml−1). (b) and (e) LDH release after 16 h. Data is expressed as mean ± SD; n = 3. (c) and (d) % YOYO-1 positive iMacs from live cell imaging taken every hour for 16 h. Data is expressed as mean ± SD; n = 3. (f) Immunoblot for FLAG-tagged DFNA5 (FLAG antibody) and caspase-3 in combined cell extract and supernatant from FLAG-tagged DFNA5 expressing iMacs 3 hrs after electroporation with flagellin (0.5 μg) and cytochrome-c (25 μg), or treated with FasL (100 ng ml−1).