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Phospho-FGFR1 (Tyr653/654) cellular kit HTRF®

The Phospho-FGFR1 kit is designed to monitor FGFR1 autophosphorylation on Tyr653 /Tyr654 as a result of FGF binding.

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  • Ease-of-use Ease-of-use
  • Highly specific Highly specific

The Phospho-FGFR1 kit is designed to monitor FGFR1 autophosphorylation on Tyr653 /Tyr654 as a result of FGF binding.

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Overview

The Phospho-FGFR1 assay is designed for a robust quantification of FGFR1 modulation, phosphorylated on Tyr653 /Tyr654, as an MAPK and PI3K/AKT pathway readout. Mutations in FGFR1 have been associated with a wide variety of cancers, including lung, breast, urothelial, or ovarian cancers.

Benefits

  • VALIDATED ON KG-1 and DMS-114 CELLS
  • SPECIFICITY

Phospho-FGFR1 (Tyr653/654) assay principle

The Phospho-FGFR1 (Tyr653/654) assay measures FGFR1 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-FGFR1 (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) assay principle

Phospho-FGFR1 (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-FGFR1 (Tyr653/654) HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

Two-plate protocol of the HTRF phospho-FGFR1 (Tyr653/654) assay

Phospho-FGFR1 (Tyr653/654) 1-plate assay protocol

Detection of Phosphorylated FGFR1 (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.

One-plate protocol of the HTRF phospho-FGFR1 (Tyr653/654) assay

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°, 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 and 4 µL of the 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 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.

Total FGFR assay validation on human cancer cell lines

Inhibition measured with Phospho (Tyr653/654) & Total-FGFR1 kits

Human KG-1 cells (bone marrow myelogenous leukaemia) were seeded in a half area 96-well culture-treated plate at 200,000 cells/well in 25 µL complete culture medium. Cells were treated with 5 µL of increasing concentrations of AZD4547, an FGFR Inhibitor, for 24h 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.

Human DMS-114 cells (lung carcinoma) were plated in a 96-well plate (100,000 cells/well) and incubated overnight. Cells were treated with a dose-response of AZD4547 for 24h at 37 °C, 5% CO2, then stimulated for 10 min with 100 ng/ml FGF2. After treatment, cells were lysed with 25µl 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-FGFR1 (Tyr653/654) or Total-FGFR1 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 FGFR1 Y653/654 phosphorylation upon treatment with AZD4547, while the FGFR1 expression level remains constant.

Validation of FGFR1 kits on KG1 and DMS-114 cells
Validation of FGFR1 kits on KG1 and DMS-114 cells

HTRF Phospho-FGFR1 (Tyr653/654) assay compared to Western Blot

KG-1 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-FGFR1 (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 8-fold more sensitive than the Western Blot, at least under these experimental conditions.

Comparison of HTRF phospho-FGFR1 (Tyr653/654) kit with Western Blot

Simplified FGFR signaling pathway

FGFRs are tyrosine kinase receptors activated by 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 FGFR phosphorylates 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.

FGFRs signaling pathway

Simplified pathway dissection with HTRF phospho-assays and CyBi-felix liquid handling

Analyse of PI3K/AKT/mTor translational control pathway - Application Notes

Cisbio lysis buffer compatibility

Cell Signaling: Biomarkers, Phospho- & total-protein Assays - Flyers

HTRF cellular phospho-protein assays

Physiologically relevant results fo fast flowing research - Flyers

Species compatibility

Cell Signaling: Biomarkers, Phospho- & total-protein assays - Flyers

Universal HTRF® phospho-protein platform: from 2D, 3D, primary cells to patient derived tumor cells

Analysis of a large panel of diverse biological samples and cellular models - Posters

HTRF phospho assays reveal subtle drug induced effects in tumor-xenografts

Tumor xenograft analysis: HTRF versus Western blot - Application Notes

HTRF cell-based phospho-protein data normalization

Valuable guidelines for efficiently analyzing and interpreting results - Application Notes

HTRF phospho-total lysis buffer: a universal alternative to RIPA lysis buffers

Increased flexibility of phospho-assays - Application Notes

Best practices for analyzing brain samples with HTRF® phospho assays for neurosciences

Insider Tips for successful sample treatment - Technical Notes

HTRF Alpha-tubulin Housekeeping kit

Properly interpret your compound effect - Application Notes

Optimize your HTRF cell signaling assays on tissues

HTRF and WB compatible guidelines - Technical Notes

Key guidelines to successful cell signaling experiments

Mastering the art of cell signaling assays optimization - Guides

HTRF phospho-assays reveal subtle drug-induced effects

Detailed protocol and direct comparison with WB - Posters

Best practices for analyzing tumor xenografts with HTRF phospho assays

Protocol for tumor xenograft analysis with HTRF - Technical Notes

How to run a cell based phospho HTRF assay

What to expect at the bench - Videos

Unleash the potential of your phosphorylation research with HTRF

Unmatched ease of use, sensitivity and specificity assays - Videos

HTRF Product Catalog 2020 July update

All your HTRF assays in one document! - Catalog

A guide to Homogeneous Time Resolved Fluorescence

General principles of HTRF - Guides

How HTRF compares to Western Blot and ELISA

Get the brochure about technology comparison. - Brochures

HTRF® cell signaling platform combined with iCell® Hepatocytes

A solution for phospho-protein analysis in metabolic disorders - Posters

Unleash the potential of your phosphorylation research with HTRF

A fun video introducing you to phosphorylation assays with HTRF - Videos

How to run a cell based phospho HTRF assay

3' video to set up your Phospho assay - Videos

Plate Reader Requirement

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