Phospho-IRF3 (Ser386) cellular kit
Fast and convenient, no wash phospho-IRF3 (Ser386) assay kit
The Mouse Total STING kit is designed to monitor the expression level of cellular mouse STING, and can be used as a normalization assay for the Mouse phospho-STING kit.
The Mouse Total STING cellular assay monitors mouse total STING, and can be used as a normalization assay with the Mouse Phospho STING kit. This kit is compatible with the buffers from the Mouse Phospho STING kit, so the same lysate can be used for analyses of both the phosphorylated and the total protein populations.
Following pathogen infection and binding of dsDNA to the cytoplasmic sensor cGAS, STING protein is phosphorylated by TBK1. This enables, its binding to IRF3 which induces IFNs type 1 production and other immune responses. The STING pathway is then switched off by STING degradation involving autophagy.
The Mouse Total STING assay quantifies the expression level of mouse STING in a cell lysate. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The Mouse Total STING assay uses two labeled antibodies, one coupled to a donor fluorophore and the other to an acceptor. Both antibodies are highly specific for a distinct epitope on the protein. In presence of STING in a cell extract, the addition of these conjugates brings the donor fluorophore into close proximity with the acceptor, and thereby generates a FRET signal. Its intensity is directly proportional to the concentration of the protein present in the sample, and provides a means of assessing the protein's expression under a no-wash assay format.
The two-plate protocol involves culturing cells in a 96-well plate before lysis, then transferring lysates to a 384-well low volume detection plate before the addition of Mouse Total STING HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.
Detection of mouse total STING with HTRF reagents can be performed in a single plate used for culturing, stimulation, and lysis. No washing steps are required. This HTS designed protocol enables miniaturization while maintaining robust HTRF quality.
Raw264.7 & NIH3T3 cells were plated under 50 µl in a 96-well plates (200,000 cells/well and 100,000 cells/well respectively) in complete culture medium and incubated overnight . The cells were stimulated with increasing concentrations of mouse specific ligand DMXAA (50µL for 1 hour). After treatment, cells were lysed with 50µl of supplemented lysis buffer #4 (1X) for 30 min at RT under gentle shaking. 16 µL of lysate were transferred into a 384 low volume white microplate before adding 4 µL of the HTRF phospho-Mouse STING or total Mouse STING detection antibodies. Finally, HTRF signals were recorded after 2 hours of incubation at RT. DMXAA induced a significant activation of the STING pathway, leading to a 8 and 6 -fold increase of STING phosphorylation in Raw264.7 and NIH3T3 cells respectively.
Raw264.7 & NIH3T3 cells were plated under 50 µl in a 96-well plates (200,000 cells/well and 100,000 cells/well respectively) in complete culture medium and incubated overnight . The cells were stimulated with increasing concentrations of c-di-AMP analog : ADU-S100 (50µL for 1 hour). After treatment, cells were lysed with 50µl of supplemented lysis buffer #4 (1X) for 30 min at RT under gentle shaking. 16 µL of lysate were transferred into a 384 low volume white microplate before adding 4 µL of the HTRF phospho-Mouse STING or total Mouse STING detection antibodies. Finally, HTRF signals were recorded after 2 hours of incubation at RT. ADUS100 induced a significant activation of the STING pathway, leading to a 9 and 5 -fold increase of STING phosphorylation in Raw264.7 and NIH3T3 cells respectively. Induced STING phosphorylation was associated with a down-regulation of its expression level, in agreement with autophagy mediated degradation.
Raw264.7 cell line was seeded in a T175 flask in complete culture medium, and incubated for 2 days at 37°C, 5% CO2. Cells were then stimulated with 100 µM DMXAA for 1 hour and lysed with 3 mL of supplemented lysis buffer #4 for 30 minutes at RT under gentle shaking. Soluble supernatants were collected after a 10-minute centrifugation. Serial dilutions of the cell lysate were performed in the supplemented lysis buffer and 16 µL of each dilution were transferred into a low volume white microplate before the addition of 4 µL of HTRF Total Mouse STING detection reagents. Equal amounts of lysates were used for a side by side comparison between HTRF and Western Blot.
A side by side comparison of Western Blot and HTRF demonstrates that the HTRF assay is 16-fold more sensitive than the Western Blot, at least under these experimental conditions.
STING, for STimulator of INterferon Genes, is a cytoplasmic homodimeric protein localized in the endoplasmic reticulum, and which plays an essential role in innate immunity. Upon pathogen infection or mitochrondrial shrinking during apoptosis, floating dsDNAs bind and activate a DNA sensor, the cyclic GMP-AMP synthase (cGAS). Activated cGAS leads to the production of 2’-3’cGAMP, a cyclic dinucleotide, which then binds to STING proteins. In turn, phosphorylated STING interacts with TANK-binding-kinase-I (TBK1), leading to the recruitment and activation of active interferon regulatory factor (IRF3) dimer. Nuclear translocation of the IRF3 dimer leads to the transcription of genes encoding IFN-α/β. In addition, the STING pathway controls NF-κB dependent inflammatory cytokine expression. As a negative feedback loop, the DNA-stimulated cGAS-STING-TBK1 pathway also triggers STING protein degradation through p62 SQSTM1 associated autophagy, to switch off IFNb production.
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