Phospho-FGFR2 (Tyr653/654) assay principle

The Phospho-FGFR2 (Tyr653/654) assay measures FGFR2 when phosphorylated at Tyr653/654. Contrary to Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis or transfer. The Phospho-FGFR2 (Tyr653/654) 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 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-FGFR2 (Tyr653/654) 2-plate assay protocol

The 2 plate protocol involves culturing cells in a 96-well plate before lysis then transferring lysates to a 384-well low volume detection plate before adding Phospho-FGFR2 (Tyr653/654) HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

Phospho-FGFR2 (Tyr653/654) 1-plate assay protocol

Detection of Phosphorylated FGFR2 (Tyr653/654) 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.

Assessment of FGFR protein levels in various human cancer cell lines

Different human cancer cell lines were seeded in T175 flasks in complete culture medium at 37°C-5% CO2. The cells were then lysed with 3 mL of supplemented lysis buffer #4 (1X) for 30 minutes at RT under gentle shaking.

25 µg of total protein for each cell line and 15 µg for KG-1 cell line were analyzed for their total FGFR1-2-3 and -4 protein levels. 16 µL of normalized samples were transferred into a 384 well low volume white microplate, and4 µL of each HTRF Total FGFR1-FGFR2-FGFR3 or FGFR4 detection antibodies were added. The HTRF signal was recorded after an overnight incubation. The results reveal a differential expression pattern for the four different FGFR receptors. Whereas FGFR1 is expressed at high levels in the DMS114 cell lung cancer model and the KG-1 bone marrow myelogenous leukaemia model, FGFR2 is preferentially expressed in SNU-16 and the Kato-III gastric cancer model, FGFR3 in the KMS-11 multiple myeloma cell line, and FGFR4 in the MDA-MD-453 breast cancer model or HuH7 hepatocarcinoma cell line. Moreover, these results demonstrate the recognition specificity provided by the HTRF Total-FGFR kits.

Inhibition measured with Phospho-FGFR2 (Tyr653/654) & Total-FGFR2 kits on SNU-16 cells

Human SNU-16 cells (gastric cancer) were seeded in a half area 96-well culture-treated plate at 100,000 cells / well in 25 µL complete culture medium. Cells were treated with 5 µL of increasing concentrations of AZD4547, an FGFR Inhibitor, for 6h at 37 ° C, 5% CO2. 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 sv white microplate, and 4 µL of the HTRF phospho-FGFR2 (Tyr653 / 654) or Total FGFR2 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 inhibition of FGFR2 Y653 / 654 phosphorylation upon treatment with AZD4547, while the FGFR2 expression level remains constant.

Inhibition measured with Phospho-FGFR2 (Tyr653/654) & Total-FGFR2 kits on Kato-III cells

Human Kato-III cells (gastric cancer) were seeded in a half area 96-well culture-treated plate at 100,000 cells / well in 25 µL complete culture medium. Cells were treated with 5 µL of increasing concentrations of AZD4547, an FGFR Inhibitor, for 6h at 37 ° C, 5% CO2. 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 sv white microplate and 4 µL of the HTRF Phospho-FGFR2 (Tyr653 / 654) or Total-FGFR2 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 inhibition of FGFR2 Y653 / 654 phosphorylation upon treatment with AZD4547, while the FGFR2 expression level remains constant.

Inhibition measured with Phospho-FGFR2 (Tyr653/654) & Total-FGFR2 kits on NCI-H716 cells

Human NCI-H716 cells (colorectal adenocarcinoma) were plated in a 96-well plate (100,000 cells/well) and incubated overnight. Cells were treated with a dose-response of AZD4547 for 6H at 37 °C, 5% CO2. After treatment, cells were lysed with 50µl 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 sv white microplate, and 4 µL of the HTRF Phospho-FGFR2 (Tyr653/654) or Total-FGFR2 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 inhibition of FGFR2 Y653/654 phosphorylation upon treatment with AZD4547, while the FGFR2 expression level remains constant.

HTRF Phospho-FGFR2 (Tyr653/654) assay compared to Western Blot

SNU-16 cells were cultured in a T175 flask in complete culture medium at 37°C, 5% CO2. After 72h incubation, the cells were 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-FGFR2 (Tyr653/654) 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.

Simplified FGFR signaling pathway

FGFRs are tyrosine kinase receptors activated by the binding of FGF ligands. This binding drives receptor homodimerization, leading to the activation of the FGFR tyrosine kinase domain and specific tyrosine residue phosphorylation. The activated receptor is a docking site for a variety of proteins that induce downstream activation of several signal transduction cascades, including the RAS-MAPK, PI3K-AKT, PLCγ, and STAT pathways. FRS2α is a key adaptor protein constitutively associated with FGFRs. The activated FGFRs phosphorylate FRS2, allowing the recruitment of GRB2 and SOS to activate RAS and the downstream RAF and MAPK pathways, particularly ERK1/2. Via GAB1, GRB2 also activates PI3K, which then phosphorylates AKT. Independently of FRS2, the binding of PLCg to the intracellular part of the activated FGFRs leads to the production of IP3 and DAG by the hydrolysis of PIP2. DAG activates the enzyme PKC, which partly reinforces the activation of the MAPK pathway. Depending on the cellular context other pathways are also activated by FGFRs, such as STAT signaling. The signals transmitted from the FGFRs to the nucleus lead to the regulation of various biological functions such as cell proliferation, differentiation, survival, adhesion, migration, and angiogenesis. Alterations of FGFRs in a wide variety of cancers are associated with the overexpression or hyperactivity of FGFRs, making the receptors key targets for anti-cancer therapies.