activates a robust protection response to gram-negative bacterias through the Defense deficiency (Imd) pathway. or in tissues lifestyle plates directly. prompted the seek out and following characterization of mammalian Toll-like receptors (5). The Drosophila Defense insufficiency (Imd) pathway stocks numerous commonalities using the TNF pathway (6). Engagement of Imd signaling is set up by the recognition of gram-negative bacterial peptidoglycan (PGN) with the Peptidoglycan Identification Protein-LC (PGRP-LC) (7-9). PGRP-LC after that indicators through the adapter proteins Imd (RIP1 ortholog) (10). This leads to the dIAP2 and Tabs2-reliant activation of dTAK1 (TAK1 ortholog) (11-13), which activates both Container (14, 15) (Bsk, JNK ortholog) as well as the Drosophila I-Kappa Kinase (IKK) complicated (16). Bsk phosphorylates and concomitantly sets off the nuclear translocation of Jra (Jun-related antigen, Jun ortholog), which companions with Indole-3-carbinol manufacture Kayak (Fos ortholog) to initiate the transcription of a subset of the immune responses genes. At the same time IKK-mediated phosphorylation of Relish (Rel, NF-kB ortholog) results in the cleavage and concomitant nuclear translocation of Rel (17, 18). While the exact mechanism of Rel processing requires clarification, the caspase-8 ortholog Dredd is essential for Rel activation and Rel processing occurs at a consensus caspase clevage site (19, 20). Thus, it appears likely that Rel is either directly cleaved by Dredd or a Dredd-responsive caspase. Cleaved Rel rapidly transcribes a large number of gene products that drive the antimicrobial response. In addition to the overt molecular similarities between the Imd and TNF pathways, there are distinct similarities between the nature of the NF-kB and JNK-mediated responses. Specifically, JNK signaling is transitory in both cases and this is due in part to NF-kB-mediated suppression Indole-3-carbinol manufacture of JNK activation. Analagous to the situation in mammals, Rel-dependent transcripts directly contribute to the inactivation of Bsk in and loss of either IKK or Rel result in enhanced Bsk signaling (21). A large number of assays exist to quantify Rel-dependent aspects of the Imd pathway; reporter constructs, Northern blot, Western blot, real-time PCR. In contrast, few protocols exist to quantify JNK activation in the Imd pathway. JNK is a member of the MAP kinase family and is activated through phosphorylation by upstream kinases. In this manuscript, we describe a protocol for the visualization and quantification of Bsk activation using commercially available ZNF346 active-JNK antibodies and infrared-based detection methods. The assay is equally applicable in Western blot analysis and for direct quantification of Bsk activation in tissue culture cells. Interestingly, we demonstrate that the In-Cell quantification method is easily extended to quantification of unrelated signaling events. Materials and Methods S2 Cell Culture S2 cells were maintained at 25C in HyQ TNM-FH medium (Gibco) supplemented with 10% heat inactivated fetal calf serum, penicillin and streptomycin. Serum free S2 cells were maintained at 25C in HyQ SFX-Insect Serum free medium (Gibco) supplemented with penicillin and streptomycin. For induction of JNK phosphorylation S2 cells were incubated in 50g/ml LPS (Sigma) at 25C for the indicated times. For induction of apoptosis S2 cells were incubated in 10M Actinomycin D (Sigma) for the indicated times. Quantification of Apoptosis For quantification of apoptosis in S2 cells by flow cytometry, 150l cells were plated into individual wells of a 96 well plate overnight at a density of 1X106 cells per ml. The cells were then incubated with 1mM CuSO4 to induce expression of Grim and fixed at various times after copper addition overnight at -20C in 70% ethanol. The cells were resuspended in PBS containing 30g/ml Propidium Iodide and 100g/ml RNase A (Both Sigma). The cells were incubated for 30min and analyzed by Indole-3-carbinol manufacture flow cytometry (FACScalibur Becton Dickinson). Western blotting and protein quantification S2 cells were harvested by centrifugation at 1000Xg for 3 minutes and lysed on ice for 10 minutes in lysis buffer Indole-3-carbinol manufacture (10mM Tris (pH 7.4), 10mM NAH2PO4, 150mM NaCl, 1% Triton X-100). Lysates were spun for 10 minutes at 21000Xg and sample loading buffer was added to the supernatants. Proteins were separated on a 10% SDS-PAGE gel and analyzed by Western blotting. Anti-phospho-JNK antibody was purchased from Cell Signaling. Anti-JNK antibody was purchased from Santa Cruz biotechnology. Alexa-fluor 680 and Alexa-fluor 750 secondary antibodies were purchased from Invitrogen. Indole-3-carbinol manufacture Protein quantification was performed using a Licor Aerius automated infrared imaging system according to manufacturer’s instructions. Plate based protein assay and quantification S2 cells were plated in 150l serum-free medium at one million cells per ml in 96 well plates and incubated for three days a 25C. Cells were fixed in 3.7% formaldehyde (Sigma) and solubilized in 0.1% Triton X-100. In Cell quantitative analysis was carried out as described in the protocol section. Briefly, fixed and permeabilized cells were blocked for one hour in blocking buffer. Afterwards, cells were incubated with anti-phospho-JNK or active caspase-3 antibodies overnight at 4C. Cells were then washed and incubated with the appropriate Alexa-fluor 750-labeled secondary antibody and alexa-fluor 680-coupled phalloidin. After a brief wash step the plates were.