Phospho-TYK2 (Tyr1054/1055) assay principle

The Phospho-TYK2 (Tyr1054/1055) assay measures TYK2 when phosphorylated at Tyr1054/1055. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The Phospho-TYK2 (Tyr1054/1055) assay uses 2 labeled antibodies: one with a donor fluorophore, the other with an acceptor. The first antibody was 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 both 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-TYK2 (Tyr1054/1055) 2-plate assay protocol

The 2 plate protocol involves culturing cells in a 96-well plate before lysis, then transferring lysates into a 384-well low volume detection plate before adding Phospho-TYK2 (Tyr1054/1055) HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

Phospho-TYK2 (Tyr1054/1055) 1-plate assay protocol

Detection of Phosphorylated TYK2 (Tyr1054/1055) 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

Phospho TYK2 (Tyr1054/1055) activation on HEL92.1.7 cells

HEL92.1.7 cells (Human erythroleukaemia) were seeded in a half area 96-well culture-treated plate at 400,000 cells / well in 25 µL complete culture medium and incubated overnight. Cells were then treated with 5 µL of increasing concentrations of Interferons for 15 minutes . After treatment, cells were lysed with 10µl of supplemented lysis buffer # 4 (4X) for 30 min at RT under gentle shaking.

After cell lysis, 16 µL of lysate were transferred into a 384-well low volume white microplate, and 4 µL of the HTRF phospho-TYK2 (Tyr1054/1055) detection reagents were added. The HTRF signal was recorded after an overnight incubation at room temperature.

As expected, the results obtained show a dose-response stimulation of TYK2 Y1054/1055 phosphorylation upon treatment with Interferons.

Phospho TYK2 (Tyr1054/1055) Inhibition on Hela cells

Hela cells (cervical cancer) were plated in 96-well culture-treated plate (200,000 cells/well) in complete culture medium, and incubated overnight at 37°C,5%CO2. Cells were treated with a dose-response of Deucravacitinib (BMS-986165) 3h at 37 °C, 5% CO2. Cells were stimulated with pervanadate 100 µM and IFN beta 1nM for 1 hour at 37°C,5%CO2. Cells were then lysed with 50 µl of supplemented lysis buffer #4 (1X) for 30 min at RT under gentle shaking. After cell lysis, 16 µL of lysate were transferred into a 384-well low volume white microplate and 4 µL of the HTRF Phospho-TYK2 (Tyr1054/1055) or Total-TYK2 detection reagents were added. The HTRF signal was recorded after an overnight incubation at room temperature.

As expected, Deucravacitinib induced a dose-dependent decrease of TYK2 phosphorylation, without effect on the expression level of the total protein.

Phospho TYK2 (Tyr1054/1055) Inhibition on HEL92.1.7 cells

HEL92.1.7 cells (Human erythroleukaemia) were seeded in a half area 96-well culture-treated plate at 400,000 cells / well in 20 µL complete culture medium and incubated overnight. Cells were treated with 5µL of a dose-response of Deucravacitinib (BMS-986165) 3h at 37 °C, 5% CO2 then with IFN beta 1nM for 15mn at 37°C,5%CO2. Cells were then lysed with 50 µl of supplemented lysis buffer #4 (1X) for 30 min at RT under gentle shaking. After cell lysis, 16 µL of lysate were transferred into a 384-well low volume white microplate and 4 µL of the HTRF Phospho-TYK2 (Tyr1054/1055) or Total-TYK2 detection reagents were added. The HTRF signal was recorded after an overnight incubation at room temperature.

As expected, Deucravacitinib induced a dose-dependent decrease of TYK2 phosphorylation, without effect on the expression level of the total protein.

HTRF Phospho TYK2 (Tyr1054/1055) Assay compared to Western Blot

Hela cells were cultured in a T175 flask in complete culture medium at 37°C, 5% CO2. After 72h incubation, the cells were first stimulated with pervanadate 100 µM and IFN alpha 2 5nM, then lysed with 3 mL of supplemented lysis buffer #4 (1X) for 30 minutes at RT under gentle shaking.

Serial dilutions of the cell lysate were performed using 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-TYK2 (Tyr1054/1055) 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 4-fold more sensitive than the Western Blot, at least under these experimental conditions.

Simplified pathway of TYK2 signaling

TYK2 in combination with JAK1, JAK2, or JAK3 interacts with different types of cytokine/interferon receptors (IL2, IL23, IFN alpha and beta). Upon cytokine activation, TYK2 and the other members of the JAK family transphosphorylate each other, and then activate the transcription factors STAT1, STAT2, STAT3, and STAT6, which dimerize. They then translocate to the nucleus to trigger the transcription of genes regulating cell differentiation, proliferation, survival, and adapted immune response.