FIG. 8.

Effects of Antcin H on the interaction between JNK and mitochondria. (A) Western blotting of Sab in mitochondria isolated from control and Antcin H treated alone at different times after APAP treatment, PHB1 is loading control. (B) Isolated liver mitochondria were incubated at 30°C for 1 h with cytoplasm obtained 2 h after APAP (300 mg/kg), with or without Antcin H (preincubated for 30 min). Extracts of washed mitochondria were then immunoblotted for p-JNK, total JNK, and loading control for mitochondria (PHB1) and cytoplasmic contamination (GAPDH). (C) Comparison of the effect of Antcin H and Dexa on p-JNK binding performed as B. Bar graphs in (B, C) represent densitometry of p-JNK/PHB1 (n = 3). *p < 0.05, **p < 0.01. (D) Mitochondria were incubated with Antcin H (25 μM) or Dexa (25 μM) in vehicle for 30 min and OCR measured as described in the Materials and Methods section in Seahorse apparatus in the presence of JNK1/2 plus ATP or p-JNK1/2 plus ATP (upper panel). The data are presented as mean ± SD of five wells for one representative experiment. Lower panel, bar graphs of oxidative phosphorylation and maximal respiration with or without Dexa and Antcin H derived from Seahorse data. Neither Dexa nor Antcin H affected OCR in the absence of p-JNK (Supplementary Fig. S2). Bar graphs summarized the results of three Seahorse experiments (oxidative phosphorylation: OCR with ADP minus oligomycin; Maximal respiration: CCCP minus AA) *p < 0.05. AA, Antimycin A; OCR, oxygen consumption rate. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars