Fig. 3. MLKL is ubiquitylated prior to plasma membrane localisation.
a Proximity ligation assay (PLA) of Mlkl−/− mouse dermal fibroblasts (MDF) reconstituted with MlklN-FLAG. Cells were treated with TNF (10 ng/ml), SM-164 (100 nM) and z-VAD-FMK (20 μM; TSZ) for 2 h. Right panel, schematic depicting the principle of PLA. Antibodies that detect MLKL and ubiquitin (Ub) were utilised. b MDFs were stimulated with TSZ for the indicated timepoints and subjected to cellular fractionation as described in the ‘Methods’ section. The corresponding cytoplasmic (C) and membrane (M) fractions were subjected to a tandem ubiquitin-binding entities (TUBE) affinity purification (AP). The experimental scheme is depicted. Prior to elution from the beads, the samples were split in two and subsequently left untreated or incubated with USP21. * refers to non-specific bands. Quantification of P-MLKL and total MLKL in the membrane fraction is shown. c Linkage-specific ubiquitin (Ub) AP in MDF stimulated with TSZ for 2 h. The indicated affinity reagents were used to purify the respective Ub chain types. * refers to a developing artefact. d Linkage-specific ubiquitin AP in HT-29 cells treated with TSZ for 6 h. The indicated affinity reagents were used to purify the respective Ub chain types. All results (a–d) are representative of those from two independent experiments. Source data are provided as a Source data file.