This HTRF kit enables the cell-based quantitative detection of phosphorylated STING as a readout of the activation of the cGAS-STING pathway

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  • No-wash
  • High sensitivity
  • All inclusive kit
  • Low sample consumption

This HTRF kit enables the cell-based quantitative detection of phosphorylated STING as a readout of the activation of the cGAS-STING pathway

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Overview

This HTRF cell-based assay conveniently and accurately quantifies phosphorylated STING at Ser366. Following pathogen infection and binding of dsDNA to the cytoplasmic sensor cGAS, STING protein is phosphorylated by TBK1, enabling its binding to IRF3 which induces IFNs type 1 production. The STING pathway is then switched off by STING degradation involving autophagy. In immuno-oncology, activating the STING pathway has shown promising anti-tumor effects in pre-clinical models and thus represents a therapeutic strategy to treat human cancer.

Phospho-STING (Ser366) assay principle

The Phospho-STING (Ser366) assay measures STING when phosphorylated at Ser366. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis or transfer. The Phospho-STING (Ser366) 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 and which 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.

Phospho-STING (Ser366) 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 adding HTRF Phospho-STING (Ser366) detection reagents. This protocol enables the cells' viability and confluence to be monitored.

Phospho-STING (Ser366) one-plate assay protocol

Detection of Phosphorylated STING (Ser366) 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.

1. 2’-3’ cGAMP induces STING phosphorylation in THP1 cells

Human THP1-R232 cells (from Invivogen) were plated under 25µl in a 96-well plates (400,000 cells/well) in complete culture medium. The cells were stimulated with increasing concentrations of 2’3’cGAMP (5µL for 4 hours). Cells were then lysed with the addition of 10 µL of supplemented lysis buffer #4 at 4X for 30 minutes 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-STING or total STING detection antibodies. In addition, Alpha-tubulin housekeeping protein was analyzed from 4 µL of cell lysate using the corresponding HTRF alpha-tubulin housekeeping kit. Finally, HTRF signals were recorded after an overnight incubation at RT. As described elsewhere, 2’3’cGAMP induced a significant activation of the STING pathway, leading to a 6-fold increase of STING phosphorylation. Induced STING phosphorylation was associated with a down-regulation of its expression level, in agreement with autophagy mediated degradation, whereas alpha-tubulin remained stable under the same conditions

HTRF Phospho and total STING enable accurate quantification of agonist-induced STING regulation.

400,000 THP1-R232 cells (from Invivogen) were plated under 25µl in 96-well plates in complete culture medium. The cells were stimulated with increasing concentrations of 2’3’cGAMP (5µL for 4 hours). Cells were then lysed with the addition of 10 µL of supplemented lysis buffer #4 at 4X for 30 minutes at RT under gentle shaking. 16 µL of lysate were analyzed either by HTRF or by Western Blot, in parallel.Signal quantification by HTRF enabled accurate statistical analysis of compound effects, as shown on the graphs below, where 400µM of cGAMP significantly decreased both total and phospho-STING (statistical method used: One way-Anova, P value < 0.0001).

HTRF phospho-STING cellular assays compared to Western Blot

The human THP1-R232 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 2’3’cGAMP for 4 hours 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-STING detection reagents. Equal amounts of lysates were used for a side by side comparison between HTRF and Western Blot.

Using the HTRF Phospho-STING assay, only 4,000 cells/well were sufficient to detect a significant signal while 63,000 cells were needed for minimal ECL signal detection using Western Blot. Therefore, in our experiment the HTRF phospho-STING assay was 16 times more sensitive than the Western Blot.

STING simplified pathway

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 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.
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