Dimerized MLKL.ND requires endogenous MLKL to induce axon degeneration. (A) Left: Representative bright-field and TMRM images of axons from DRG neurons expressing MLKL.ND 24 h after BB addition. Neurons express either control or MLKL targeted shRNA. The MLKL shRNA targets the C-terminal half of MLKL and therefore does not recognize MLKL.ND. Scale bar, 50 µm. Right: Quantitative analysis of axon degeneration at the indicated times after BB application for the indicted conditions. Axons expressing shRNA to endogenous MLKL are protected for at least 48 h after dimerization of MLKL.ND. Axons with a DI >0.3 are defined as degenerated (dashed line). Data represent the mean ± SEM, n = 4; two-way ANOVA with post hoc Bonferroni’s multiple comparison test; time, F(1.116, 6.695) = 159.4; *, P < 0.05; **, P < 0.01; and ***, P < 0.001. (B) Left: Immunoblot of phosphorylated MLKL in lysates from neurons expressing MLKL.ND collected at 0 or 8 h after BB addition. TUJ1 is used as a loading control. Dimerization of MLKL.ND triggers phosphorylation of endogenous MLKL. Right: Quantification of phosphorylated MLKL levels normalized to TUJ1. Data represent the mean ± SEM, n = 3; two-tailed unpaired t test, P = 0.0283; *, P < 0.05; **, P < 0.01; and ***, P < 0.001. (C) Immunoblot analysis as in B but including necrostatin-1s addition where indicated. This experiment demonstrates that treatment with the RIPK1 inhibitor necrostatin-1s does not prevent the phosphorylation of MLKL induced by dimerization of MLKL.ND. These data in conjunction with Fig. S3 demonstrate that MLKL.ND does not require upstream RIPK1.