Jnk inhibitor 9

Succinimidyl ester conjugated to Alexa Fluor 647 and DAPI were purchased from Life Technologies (Burlington, ON). The following small molecule inhibitors were purchased from Selleckchem (Houston, TX): Rapamycin, Torin2, SB203580, VX-702, SB202190 (FHPI), BIRB 796 (Doramapimod), FR 180204, PD184352 (CI-1040), PD98059, JNK Inhibitor II, JNK Inhibitor IX. Lentiviral expression vectors encoding the JNK and p38 MAPK Kinase Translocation Reporters (KTR) were a kind gift from Markus Covert (Addgene plasmids No. 59151 and 59155). ON-TARGETplus SMARTpool siRNAs for the genes of interest as well non-targeting negative control siRNAs were obtained from Dharmacon (Lafayette, CO). Phospho-p38 MAPK (Thr180/Tyr182) antibody (RRID:AB_2139682) for immunofluorescence was purchased from Cell Signaling (Beverly, MA).

The imidazole drug SB202190 (Sigma-Aldrich) was added to standard fly food to prevent p38 activation. We used three different concentrations (0.12 μM, 1 μM and 5 μM), and DMSO as the control. To inhibit chemically JNK we used the JNK Inhibitor IX (5 μM, Selleckchem) which is a thienylnaphthamide compound that is a selective and potent inhibitor of the ATP binding site of JNK. The timing and protocol followed to inhibit both pathways was the same as that to scavenge ROS.