Necroptosis is emerging as a critical pathogenic mechanism in several liver diseases, including cholestatic disorders. Necroptosis was recently described as a novel cell death subroutine, activated downstream of death receptor stimulation and dependent on receptor-interacting serine/threonine-protein kinase 3 activity and mixed lineage kinase domain-like oligomerization and translocation to cell membrane. Here, we describe a combination of methods to evaluate necroptosis triggering in in vitro and in vivo models of cholestasis. Particularly, we detail alternative protocols to isolate total and soluble/insoluble protein extracts from tissues and cell cultures, as well as in vitro receptor-interacting serine/threonine-protein kinase 3 kinase activity assays, and subsequent Western blot analysis.
Keywords: Cell death; Cell signaling; Cholestasis; Insoluble protein fractions; Mixed lineage kinase domain-like (MLKL); Necroptosis; Necrosome; Receptor-interacting serine/threonine-protein kinase 3 (RIPK3); Tumor necrosis factor-α (TNF-α); Western blot.