Fig. 1: Mb27 binds activated human MLKL.

a Wild-type (WT) HT29 cells expressing Mb27 or Mb32 following doxycycline (Dox) induction were untreated (UT) or treated with the necroptotic stimulus, TSI (TNF; Smac mimetic, Compound A; and pan-Caspase inhibitor, IDN-6556), for 3 h and monobodies immunoprecipitated via their N-terminal FLAG-tag. Mb32 coimmunoprecipitated MLKL in both presence and absence of TSI stimulation, whilst Mb27 coimmunoprecipitated MLKL only in the presence of TSI. b Mb27 immunoprecipitated MLKL from lysates of RIPK3^−/− HT29 cells in the absence and presence of necroptotic stimulation (TSI, 3 h). c Wild-type HT29 cells were treated with a necroptotic stimulus (TSI) over a 3 h timecourse, and Mb27 immunoprecipitation of MLKL examined. Mb27 co-immunoprecipitation of MLKL followed the emergence of RIPK3-mediated MLKL S358 phosphorylation (pMLKL) 1.5 h post-necroptotic stimulation. All images and blots are representative of at least two independent experiments. d FLAG-immunoprecipitates of Mb27 or Mb32 expressed (+Dox) in wild-type HT29 cells ±7.5 h TSI treatment revealed the presence of pRIPK3 bound to MLKL, with Mb27 immunoprecipitating MLKL only after TSI-stimulation. A representative of 4 independent experiments is shown. e FLAG-human RIPK3 expressed (+Dox) in RIPK3^−/− HT29 cells coimmunoprecipitates MLKL in the presence or absence of TSI-stimulation, but not pMLKL post-TSI treatment. Lysates and IP samples were run on the same gel and subjected to the same exposure. A representative of independent duplicates is shown in each of (c, e). f Recombinant His₆-human MLKL pseudokinase domain and RIPK3 kinase domain complex expressed and purified from insect cells was untreated (UT) or incubated with combinations of Mg²⁺, ATP or AMPPNP, the complex trapped by Ni-NTA chromatography and the eluates probed by immunoblot for RIPK3 and MLKL. Only ATP/Mg²⁺ induced dissociation of RIPK3 and MLKL. Triplicates are shown for each condition.