C57BL/6 and Ripk3^–/– mice were i.v. injected with ConA and analyzed 7 hours later. Experiments were repeated 3 times with similar results. (A) Representative images of liver tissue sections double stained for RIPK3 and β-catenin (confocal images are on the right; the dashed line separates necrotic areas from nonnecrotic areas; arrows demonstrate RIPK3-positive immune cells; asterisks mark representative hepatocytes). (B) Plasma AST concentrations of ConA-challenged control and Ripk3^–/– mice (n > 3 per group) and evaluation of necrotic areas in TUNEL assay–stained liver sections of control and Ripk3^–/– mice (from C, n > 3 per group). (C) Representative images of immunohistochemical (TUNEL assay) staining analysis of hepatic tissue sections. (D) Representative images of MLKL-stained liver tissue sections. (E) Quantification of plasma membrane localized MLKL (from D, n > 4 per group). (F) Western Blot analysis demonstrating that endogenous MLKL locates at the plasma membrane (PM) in Ripk3^–/– mice following ConA treatment. C; cytoplasm. (G) Endogenous MLKL was immunoprecipitated with anti-MLKL antibody in lysates of L929 cells (treated for 3 hours with TNF-α/zVAD/SMAC mimetic) or in liver lysates of ConA-challenged wild-type or Ripk3^–/– mice. (H) Recombinant hRIPK3, but not hRIPK1, kinase domain undergoes autophosphorylation and can mediate hMLKL phosphorylation in vitro. Phosphoryl transfer of ³²P by 25 ng/μl hRIPK1 or hRIPK3 kinase domain in the absence of hMLKL, or 75 ng/μl hMLKL in the absence of kinase, or 2.5 ng/μl hRIPK1 or hRIPK3 in the presence of 75 ng/μl hMLKL was detected by autoradiography. A Coomassie-stained image of the autoradiograph is shown. The images are representative of triplicate experiments. Error bars indicate +SD, gene expression levels are shown relative to HPRT/Hprt. Scale bar: 5 μm (A, right, and D, right); 50 μm (A, left, and D, left); 250 μm (C); magnification in (C); ×11.