Fig. 3. MLKL-KLD dimerization mutant blocks necroptosis by preventing MLKL monomer-oligomer transition.

A MLKL KO HT-29 cells stably expressing flag-tagged wild-type (WT) or dimerization mutant (AAAGAA) of MLKL were treated with DMSO or T/S/Z combine with MLKL inhibitor 2 μM Necrosulfonamide (NSA) for 12 h. Then, the cells were harvested and whole-cell lysates were prepared and subjected to SDS-PAGE followed by western blot analysis using anti-phosphor-MLKL and anti-Flag antibodies. The expression of β-Actin was shown as a loading control. B Gel filtration analysis of purified recombinant proteins of His-Sumo tagged human MLKL (126~181 aa) containing the internal coiled-coil region on Superdex 200. The elution fractions were applied to SDS-PAGE followed by Coomassie blue staining. The elution positions of size standards are indicated (Thymoglobulin, 670 kDa; Ferritin, 440 kDa; Aldolase, 75 kDa; Carbonic Anhydrase, 29 kDa). C Plasmids containing Wild-type (WT), KLD-dimer mutant (AAAGAA), or coiled-coil mutant(4 A) were transfected transiently into HEK293T cells with stable-expressed human RIPK3 (HEK293T-RIPK3) for about 12 h in 60 mm dishes. Then the cells were treated with T/S/Z for 8 h and harvested. And the whole-cell lysates were mixed with DMSO or crosslinker DSG as described in the MATERIALS AND METHODS. The samples were analyzed by western blotting using an anti-Flag (MLKL).