This Total DDR1 assay quantifies the expression level of DDR1 in a cell lysate. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The Total DDR1 assay uses two labeled antibodies, one coupled to a donor fluorophore and the other to an acceptor. Both antibodies are highly specific for a distinct epitope on the protein. In the presence of DDR1 in a cell extract, the addition of these conjugates brings the donor fluorophore into close proximity with the acceptor and thereby generates a FRET signal. Its intensity is directly proportional to the concentration of the protein present in the sample, and provides a means of assessing the protein's expression 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 HTRF Total DDR1 detection reagents. This protocol enables the cells' viability and confluence to be monitored.

Detection of Total DDR1 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 (Tyr796, Tyr513 or panTyr) or Total DDR1 detection reagents were added. The HTRF signal was recorded after an overnight incubation.

As expected, the total DDR1 assay showed a constant level of Total DDR1 under Pervanadate, whereas the treatment triggered a dose-dependent increase in the level of Phospho-DDR1.

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 Wild Type 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 and Phospho (panTyr), 1/5 for Phospho (Tyr796) 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 (Tyr796, Tyr513 or panTyr) or Total DDR1 detection reagents were added. The HTRF signal was recorded after an overnight incubation.

As expected, the total DDR1 assay showed a constant level of Total DDR1 under Pervanadate or collagen, whereas the treatment triggered a dose-dependent increase in the level of Phospho-DDR1.

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 doses 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 (Tyr796, Tyr513 or panTyr) 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 Wild Type using DharmaFECT kb (Horizon Discovery). After 24h of incubation, the cells were treated for 2 hours with increasing doses of Dasatinib, 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 and Phospho (panTyr), 1/5 for Phospho (Tyr796) 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 (Tyr796, Tyr513 or panTyr) 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, 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, DDR1ba, 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 Total DDR1 detection antibodies were added. The HTRF signal was recorded after an overnight incubation.

Cell transfection with DDR1a and DDR1b led to the detection of the receptor compared to the negative control (untransfected). On the contrary, the transfection of DDR2 did not induce any signal increase, demonstrating that the HTRF Total DDR1 assay is selective for DDR1a and DDR1b and 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 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 Total DDR1 detection reagents. The HTRF signal was recorded after an overnight incubation.

The HTRF Total DDR1 assay efficiently detected Total DDR1 in various cellular models expressing different levels of the protein.

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 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 Total DDR1 detection reagents. Equal amounts of lysates were used for a side-by-side comparison between HTRF and Western Blot.

Using the HTRF Total DDR1 assay, 20 cells/well were enough to detect a significant signal, while 680 cells were needed to obtain a minimal chemiluminescent signal using Western Blot. Therefore, in these conditions, the HTRF Total DDR1 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.