Phospho-RET (pan) assay principle

The Phospho-RET (pan) assay measures RET when phosphorylated on Tyrosines. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The assay uses 2 antibodies, one labeled with a donor fluorophore and the other with an acceptor. The first antibody is selected for its specific binding to phosphorylated tyrosine residues, the second for its ability to recognize specifically the protein independently 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-RET (pan) two-plate assay protocol

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

Phospho-RET (pan) one-plate assay protocol

Detection of Phosphorylated RET (pan) 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.

Inhibition of RET phosphorylation in mouse neuroblastoma Neuro-2a cells

The mouse neuroblastoma cell line Neuro-2a was seeded in a 96-well culture-treated plate under 100,000 cells/well in complete culture medium, and incubated overnight at 37 °C, 5% CO2. The cells were treated for 2 hours with increasing doses of Pralsetinib, and 100 µM Pervanadate were added 30 minutes before the end of the treatment. The cells were lysed with 50 µL of supplemented lysis buffer #4 for 30 minutes at RT under gentle shaking. For the detection step, 16 µL of cell lysate were transferred into a 384-well low volume white microplate and 4 µL of the HTRF Phospho-RET (pan) or Total-RET detection reagents were added. The HTRF signal was recorded after an overnight incubation.

As expected, the RET kinase inhibitor Pralsetinib induced a dose-dependent decrease in RET phosphorylation, without effect on the expression level of the receptor.

Inhibition of RET phosphorylation in the human lung adenocarcinoma cell line LC-2/ad

The human lung adenocarcinoma cell line LC-2/ad was seeded in a 96-well culture-treated plate under 50,000 cells/well in complete culture medium, and incubated overnight at 37 °C, 5% CO2. The cells were treated for 2 hours with increasing doses of Pralsetinib, and 100 µM Pervanadate were added 30 minutes before the end of the treatment. The cells were lysed with 50 µL of supplemented lysis buffer #4 for 30 minutes at RT under gentle shaking. For the detection step, 16 µL of cell lysate were transferred into a 384-well low volume white microplate and 4 µL of the HTRF Phospho-RET (pan) or Total-RET detection reagents were added. The HTRF signal was recorded after an overnight incubation.

As expected, the RET kinase inhibitor Pralsetinib induced a dose-dependent decrease in RET phosphorylation, without significant effect on the expression level of the receptor.

RET signaling pathway

RET is activated by Glial cell line-derived neurotrophic Family Ligands (GFL) including GDNF (glial cell line-derived neurotrophic factor), NRTN (neurturin), ARTN (artemin) and PSPN (persephin). Ligand binding to GDNF Family Receptor-α co-receptors (GFRα1/2/3/4) triggers the formation of a complex with RET, leading to RET dimerization and autophosphorylation on multiple intracellular tyrosines. Phosphorylated tyrosine residues serve as docking sites for various adaptor proteins that induce the activation of downstream signaling pathways (MAPK, PI3K/AKT, STAT3...) necessary for cell survival, differentiation, proliferation and motility.