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. 2017 Jun 12;18(8):1429–1441. doi: 10.15252/embr.201743947

Figure EV4. Mutation of DAI's RNA binding domain delays necroptosis, and human DAI can mediate necroptosis.

Figure EV4

  1. Immunoprecipitation (IP) and IB analysis to detect FLAG‐DAI/ZBP1, myc‐RIPK3, and β‐actin from 293T cells transfected with vectors expressing 6myc‐RIPK3 and different 3XFLAG‐DAI/ZBP1 constructs, and subjected to IP with anti‐FLAG M2 agarose beads.
  2. Cell death of DAI/ZBP1‐reconstituted 29‐11 cell lines infected with M45mutRHIM MCMV (MOI = 5); 6, 8, 10, and 12 h post‐infection times were extracted from Fig 5D–F and replotted for clarity. ****P < 0.0001; ***P < 0.001; *P < 0.05; n.s., not significant (P > 0.05) by one‐way ANOVA with Dunnett's multiple comparisons test, compared to EV. Error bars indicate standard deviation from the mean (SD; n = 4 biological replicates).
  3. Relative viability of HT‐29 cells with or without hDAI expression infected with M45mutRHIM compared to WT MCMV. ***P < 0.001 by two‐tailed unpaired Student's t‐test. Error bars indicate SD (n = 3 biological replicates).
  4. IB analysis to detect p‐MLKL, total MLKL, and β‐actin from HT‐29 cells with or without hDAI expression infected with WT or M45mutRHIM for 10 h.
  5. Relative viability of 29‐11 cells reconstituted with empty vector (EV) or DAI‐ΔZα1mutZα2 infected with M45mutRHIM compared to WT MCMV. ***P < 0.001 by two‐tailed unpaired Student's t‐test. Error bars indicate SD (n = 3 biological replicates).