Inhibition measured with Phospho-JAK2 (Tyr1007/1008) & Total-JAK2 kits on HEL92.1.7 cell

HEL92.1.7 cells (Human erythroleukaemia) were seeded in a half area 96-well culture-treated plate at 200,000 cells / well in 20 µL complete culture medium. Cells were treated with 5 µL of increasing concentrations of Ruxolitinib (type I JAK inhibitor) or CHZ868 (Type II JAK inhibitor) for 1h at 37 ° C, 5% CO2 followed by a stimulation step with 5 µL of pervanadate 100 µM during 30 minutes . After treatment, cells were lysed with 10 µl of supplemented lysis buffer # 1 (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-JAK2 (Tyr1007/1008) or Total JAK2 detection reagents were added. The HTRF signal was recorded after 3 hours at room temperature.

As expected, Ruxolitinib or CHZ868 inhibitors induced a dose-dependent decrease in JAK2 phosphorylation, without effect on the expression level of the total protein.

 

 

Inhibition of JAK2 phosphorylation (Tyr1007/1008) on THP1 cells

THP1 cells (Human monocytic leukemia ) were seeded in a half area 96-well culture-treated plate at 400,000 cells / well in 20 µL complete culture medium. Cells were treated with 5 µL of increasing concentrations of Ruxolitinib, Tofacitinib or Pacritinib for 1h at 37 ° C, 5% CO2 followed by a stimulation step with 5 µL of pervanadate at 100 µM during 30 minutes . After treatment, cells were lysed with 10 µl of supplemented lysis buffer # 1 (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-JAK2 (Tyr1007/1008) or Total JAK2 detection reagents were added. The HTRF signal was recorded after 3 hours at room temperature.

As expected, the results obtained show a dose-response inhibition of JAK2 Y1007/1008 phosphorylation upon treatment with Ruxolitinib, Tofacitinib or Pacritinib, while the JAK2 expression level remains constant.

 

 

 

Phospho -JAK2 (Tyr1007/1008) down-regulation by siRNA

HEL92.1.7 cells were treated with 2.5 µM of Accell siRNA (Horizon) targeting specifically JAK1, JAK2, JAK3 or with a non-targeting siRNA (included as control), in a 96-well plate (200,000 cells/well) under 100 µL. After 48h incubation at 37°C, 50 µL of complete culture medium was added and the cells were incubated for an additional 24h-incubation at 37°C. Cells were stimulated with Pervanadate (100 µM) during 30 minutes and after plate centrifugation, were lysed with 50 µL of supplemented lysis buffer #1 (1X) and 16 µL of lysates were transferred into a low volume white microplate before the addition of 4 µL of premixed HTRF phospho-JAK2(Tyr1007/1008) detection antibodies. The HTRF signal was recorded after an overnight incubation at RT.

Cell treatment with JAK2 siRNA led to a significant downregulation of phospho JAK2 (Tyr1007/1008) with 55% signal decrease compared to the cells transfected with the non-targeting siRNA. No decrease of signal was observed with cells treated with JAK1 or JAK3 siRNA , demonstrating the specificity of the kit

 

HTRF Phospho-JAK2 (Tyr1007/1008) assay compared to Western Blot

HEL92.1.7 cells were cultured in a T175 flask in complete culture medium at 37°C, 5% CO2. After 72h incubation, the cells were first stimulated with pervanadate 100 µM for 30 minutes, then lysed with 3 mL of supplemented lysis buffer #1 (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-JAK2 (Tyr1007/1008) 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.