Fig. 3.

Mitoneet mediates the TNFα-induced translocation of Stat3–Grim-19 to the mitochondria. (A) Hepatocytes treated with siRNA targeting microneet or a non-targeting control siRNA (as in Fig. 2A) were pre-treated or not for 30 minutes with 10 µM of necrostatin and then treated with 10 ng/ml of TNFα. After 4 hours of TNFα treatment, the hepatocytes were harvested and mitochondria isolated. Mitochondrial extracts were prepared, separated by SDS-PAGE and then transferred to PVDF membranes. The blots were probed with antibodies specific for Grim-19, STAT3 or mitoneet. The blots were then stripped and re-probed with antibodies against VDAC-1. The results are representative of three independent experiments. (B) Mitoneet was immunoprecipitated from mitochondrial lysates from hepatocytes treated as in A. The immunoprecipitates were then separated by SDS-PAGE and blotted onto PVDF membranes. The blots were probed with antibodies against Grim-19 or STAT3. For the reverse immunoprecipitation, Grim-19 was immunoprecipitated from mitochondrial lysates and the immunoprecipitates separated by SDS-PAGE and blotted onto PVDF membranes. The blots were probed with antibodies against mitoneet or STAT3. The results are representative of three independent experiments. (C) Hepatocytes were transfected with siRNA targeting Grim-19 or Stat3 and then treated as in A. At the time points indicated, the cells were harvested and viability determined by uptake of propidium iodide. Values are the means ± s.d. of three independent experiments.