Upon regulated necrosis, receptor interacting protein kinase 3 (RIPK3) phosphorylates the mixed lineage kinase domain-like (MLKL) protein at Thr357 and Ser358 located in the activation loop of its pseudokinase domain. The RIPK3-mediated phosphorylation relieves the inhibitory effect of the pseudokinase domain of MLKL, thus allowing the activated MLKL to oligomerize and translocate from the cytosol to cell membranes to cause membrane disintegration. The flotillin-mediated endocytosis was found to antagonize MLKL-mediated plasma membrane alteration to sustain survival of the cell (Fan W et al. 2019). Protein cross-linking followed by affinity purification assays detected the lipid raft-associated proteins flotillin-1 (FLOT1) and flotillin-2 (FLOT2) in membrane-localized MLKL immunoprecipitates isolated from human colon cancer HT-29 cells expressing MLKL fused with a 3xFlag-HA (in which MLKL fused with a 3xFlag-HA (hemagglutinin) tagged to its C terminus was expressed while endogenous MLKL was knocked down (Fan W et al. 2019). Mass spectrometry and immunoblotting assay identified showed that MLKL was associated with FLOT1 and FLOT2 within extracellular vesicles (EVs) released from caspase inhibitor z-VAD-fmk-treated HT-29 cells, suggesting that RIPK3 activation triggered MLKL association with flotillins (Yoon S et al. 2017). Phosphorylated MLKL was eventually directed to lysosomes in HT-29 cells immunolabeled with phospho-MLKL and LAMP1 (lysosome-associated membrane protein 1) antibodies (Fan W et al. 2019). Bafilomycin A1 prevented the degradation of phosphorylated MLKL only in parental HT-29 cells but not in cells lacking either flotillin suggesting that flotillins mediated the relocalization of phospho-MLKL from cell membranes to lysosomes (Fan W et al. 2019). However, the other study reported that phosphorylated MLKL did not colocalize to a significant extent with LAMP-2-containing lysosomes, and inhibiting lysosomal degradation during the effector phase of necroptosis did not significantly alter the extent of necroptotic cell death (Samson AL et al. 2020). Binding between the N-terminal helix bundle of phospho-MLKL and a C-terminal region of flotillin-1 was required for flotillin-mediated endocytosis of phosphorylated MLKL (Fan W et al. 2019). In addition, mice with a double knockout of Flot1 and Flot2 showed accelerated death upon injection with TNFα plus z-VAD-fmk to induce systemic inflammatory response syndrome (SIRS). Moreover, all flotillin-null mice survived TNFα/z-VAD-fmk-induced SIRS when coinjected with the RIPK1 inhibitor RIPA-56, confirming that the accelerated death in flotillin-null mice resulted from enhanced necroptosis (Fan W et al. 2019). Thus, phosphorylated MLKL is removed from membranes through FLOT1, 2-mediated endocytosis followed by lysosomal degradation (Fan W et al. 2019).