Mouse Phospho-STING (Ser365) assay principle

The Mouse Phospho-STING (Ser365) assay measures mouse STING when phosphorylated at Ser365. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The Mouse Phospho-STING (Ser365) assay uses 2 labeled antibodies: one with a donor fluorophore, the other one with an acceptor. The first antibody is selected for its specific binding to the phosphorylated motif on the protein, the second for its ability to recognize the protein independent of its phosphorylation state. Protein phosphorylation enables an immune-complex formation involving the two labeled antibodies. This brings the donor fluorophore into close proximity to the acceptor, thereby generating a FRET signal. Its intensity is directly proportional to the concentration of phosphorylated protein present in the sample, and provides a means of assessing the protein's phosphorylation state under a no-wash assay format.

Mouse Phospho-STING (Ser365) two-plate assay protocol

The two-plate protocol involves culturing cells in a 96-well plate before lysis, then transferring lysates to a low volume detection plate (either HTRF 384-lv or 96-lv plate) before the addition of HTRF Mouse Phospho-STING (Ser365) detection reagents. This protocol enables the cells' viability and confluence to be monitored.

Mouse Phospho-STING (Ser365) one-plate assay protocol

Detection of Mouse Phosphorylated STING (Ser365) 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.

3’3’cGAMP induces Mouse STING phosphorylation in Raw264.7 cells

Raw264.7 cells were plated under 50 µl in a 96-well plates (200,000 cells/well) in complete culture medium and incubated overnight . The cells were stimulated with increasing concentrations of bacterial cyclic di-nucleotide ligand : 3'3'cGAMP (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. 3'3'cGAMP induced a significant activation of the STING pathway, leading to a 5 -fold increase of STING phosphorylation.

HTRF phospho- Mouse STING cellular assay compared to Western Blot

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 phospho- 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 both technology give the same sensitivity under these experimental conditions.

STING simplified pathway

STING, for STimulator of INterferon Genes, is a cytoplasmic homodimeric protein localized in the endoplasmic reticulum, 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, switching off IFNβ production.