The Phospho-DDR1 (Tyr513) assay measures DDR1 when phosphorylated at Tyr513. 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 was selected for its specific binding to the phosphorylated motif on the protein, and the second for its ability to recognize 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.

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-DDR1 (Tyr513) HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

Detection of Phosphorylated DDR1 (Tyr513) 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.

T-47D cells were seeded in a 96-well culture-treated plate (100,000 cells/well) in complete culture medium, and incubated overnight at 37°C, 5% CO2. The cells were treated for 30 minutes with increasing concentrations of Pervanadate.

After 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-DDR1 (Tyr513) or Total DDR1 detection reagents were added. The HTRF signal was recorded after an overnight incubation.

As expected, Pervanadate triggered a dose-dependent increase in the level of Phospho-DDR1 (Tyr513).

HEK293 cells were seeded in a 96-well culture-treated plate (100,000 cells/well) in complete culture medium, and incubated overnight at 37°C, 5% CO2. The cells were transfected with DDR1b using DharmaFECT kb (Horizon Discovery). After 24h of incubation, the cells were treated with increasing concentrations of Collagen (from C1 to C3: 0.625, 1.25, and 10 µg/mL) for 1h or Pervanadate for 30 minutes (100 µM).

After 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, cell lysates were diluted 1/20 for Total or ½ for Phospho (Tyr513) and then 16 µL were transferred into a 384-well low volume white microplate. 4 µL of the HTRF Phospho-DDR1 (Tyr513) or Total DDR1 detection reagents were added. The HTRF signal was recorded after an overnight incubation.

As expected, Collagen (Endogeneous ligand of DDR1 receptor) triggered a dose-dependent increase in the level of Phospho-DDR1 (Tyr513), as did Pervanadate.

T-47D cells were seeded in a 96-well culture-treated plate (100,000 cells/well) in complete culture medium, and incubated overnight at 37°C, 5% CO2. The cells were treated for 4 hours with increasing concentrations of Dasatinib, 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-DDR1 (Tyr513) or Total-DDR1 detection reagents were added. The HTRF signal was recorded after an overnight incubation.

As expected, the DDR1 kinase inhibitor Dasatinib induced a dose-dependent decrease in DDR1 phosphorylation, but also a slight inhibition in the expression level of the receptor, while no toxicity was detected (ATPlite Luminescence Assay System, #6016943).

HEK293 cells were seeded in a 96-well culture-treated plate (100,000 cells/well) in complete culture medium, and incubated overnight at 37°C, 5% CO2. The cells were transfected with DDR1b using DharmaFECT kb (Horizon Discovery). After 24h of incubation, the cells were treated for 2 hours with increasing concentrations of DDR1-IN-1, and 10 µg/mL Collagen were added 1h 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, cell lysates were diluted 1/20 for Total or ½ for Phospho (Tyr513), and then 16 µL were transferred into a 384-well low volume white microplate. 4 µL of the HTRF Phospho-DDR1 (Tyr513) or Total-DDR1 detection reagents were added. The HTRF signal was recorded after an overnight incubation.

As expected, the DDR1 kinase inhibitor DDR1-IN-1 induced a dose-dependent decrease in DDR1 phosphorylation, without any effect on the expression level of the receptor.

HEK293 cells were plated in a 96-well plate (25,000 cells/well) and cultured for 24h. The cells were then transfected with different plasmids, DDR1a, DDR1b, or DDR2, as well as with a negative control. Following a 24h incubation, cells were treated with Pervanadate (100 µM, 30 min).

After cell lysis, 16 µL of lysates were transferred into a 384-well low volume white microplate and 4 µL of the HTRF Phospho-DDR1 (Tyr513) detection antibodies were added. The HTRF signal was recorded after an overnight incubation.

Cell transfection with DDR1b only led to the detection of the Phospho-DDR1 (Tyr513) compared to the negative control. On the contrary, the transfection of DDR1a or DDR2 did not induce any signal increase, demonstrating that the Phospho-DDR1 (Tyr513) assay is specific for DDR1b and as expected, does not cross-react with a DDR2 family member.

Adherent (MCF7 & A431) and suspension (K562) human cells were seeded at respectively 100,000 and 200,000 cells/well in a 96-well microplate. After 24h of incubation, the cells were treated for 30 min with 100 µM of Pervanadate before being lysed for 30 minutes with supplemented lysis buffer #4, following the protocol for adherent or suspended cells, at RT under gentle shaking.

16 µL of lysate were transferred into a 384-well low volume white microplate before the addition of 4 µL of the HTRF Phospho-DDR1 (Tyr513) detection reagents. The HTRF signal was recorded after an overnight incubation.

The HTRF Phospho-DDR1 (Tyr513) assay detected DDR1 phosphorylation at residue Y513 in various cellular models with different phosphorylation levels.

HEK293 cells were grown in a T175 flask in complete culture medium at 37°C - 5% CO2 until 80% confluence. Transfection of DDR1b was performed with DharmaFECT kb (Horizon Discovery). After 24h of incubation, the cells were treated for 30 min with 100 µM of Pervanadate before being 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-DDR1 (Tyr513) detection reagents. Equal amounts of lysates were used for a side-by-side comparison between HTRF and Western Blot.

Using the HTRF Phospho-DDR1 (Tyr513) assay, 80 cells/well were enough to detect a significant signal, while 2710 cells were needed to obtain a minimal chemiluminescent signal using Western Blot. Therefore, in these conditions, the HTRF Phospho-DDR1 (Tyr513) assay was 32 times more sensitive than the Western Blot technique.

DDR1 is activated by collagen binding that triggers its dimerization into a complex that in turn autophosphorylates on multiple intracellular tyrosines. Phosphorylated tyrosine residues serve as docking sites for various adaptor proteins. These induce the activation of downstream signaling pathways (MAPK, PI3K/AKT, STAT3, Pyk2, or TRAF6...) necessary for cell proliferation, adhesion, migration, and ECM remodeling.