Both Bax and Bim activation resulted in mitochondrial translocation triggering the intrinsic death pathway. ConA or GalN/LPS stimulation resulted in activation of Bax and Bim that

was inhibited by TAT-ARC pretreatment (Fig. 6C). TAT-ARC application abrogated Bim mitochondrial translocation following ConA or GalN/LPS stimulation (data not shown) but no interaction of ARC and Bim was detected (data not shown). However, due to FXR agonist the direct ARC-Bax interaction it remains unclear whether abrogated Bax activation results from ARC’s inhibition of Bax or JNK only or a combination of both. Thus, our results suggest that abrogated Bax activation might result from direct inhibition by ARC or, alternatively, from ARC-mediated JNK inhibition, whereas impaired Bim activation is most likely an indirect effect of ARC, probably mediated through JNK inhibition. The pathophysiological relevance of JNK signaling in TNF-mediated models of ALF was demonstrated in mice treated with the small molecule JNK inhibitor, SP600125, showing JNK-dependent survival (Fig. 6D). These observations clearly show that JNK signaling is critically involved in mediating hepatotoxicity in both models. Our results demonstrated that in both models of TNF-dependent liver

injury ARC-dependent protection is associated with JNK inhibition. Hence, we sought whether ARC/JNK interaction might be involved in mediating protection, and thus performed immunoprecipitation experiments to test this hypothesis. Immunoprecipitation of lysates from TAT-ARC-transduced selleck livers demonstrated binding of TAT-ARC to endogenous JNK1 and JNK2, respectively (Fig. 7A). The interactions of ectopic ARC with both JNK1 and JNK2 were further confirmed using JNK1 and JNK2-specific antibodies (Fig. 7B). To exclude unspecific antibody binding, because eight JNK isoforms exist at the messenger RNA level, and

to investigate whether interactions Terminal deoxynucleotidyl transferase between ARC and JNK are direct or indirect, a cell-free system was used (Fig. 7C). Applying a cell-free system with both recombinant JNK1 and JNK2 protein proved the specificity of ARC JNK1 and JNK2 interactions. Furthermore, our results demonstrated that ARC interacts directly with JNK1 and JNK2 (Fig. 7C). Although TAT-ARC interacted with JNK1 and JNK2, it did not bind other relevant mediators of TNF signaling such as Flip, RIP, TRADD, or TRAF2 (data not shown). These results suggest that ectopic ARC protein inhibits JNK activation and translocation in vivo by binding to endogenous JNK1 and JNK2 in the liver. To elucidate the physiological occurrence of the ARC-JNK interaction, immunoprecipitations were performed using murine heart and skeletal muscle lysates that express ARC, JNK1, and JNK2 endogenously.7 Immunoprecipitation experiments confirmed interactions of endogenous ARC with endogenous JNK1 and JNK2 in skeletal muscle (Fig. 7D).