Analysis of TBK1 phosphorylation to monitor the LPS/TLR4 axis in Kupffer cells

Mouse Kupffer cells (ImKC cell line) were plated in 96-well plates (200,000 cells/well) in complete culture medium and incubated at 37°C - 5% CO2. The day after, cells were treated for 1 hour with increasing concentrations of LPS diluted in serum-free medium. After medium removal, cells were then lysed with 50 µL of supplemented lysis buffer #4 for 30 minutes at RT under gentle shaking, and 16 µL of lysate were transferred twice over into a low volume white microplate before adding 4 µL of the HTRF® phospho-TBK1 or total TBK1 detection antibodies. The HTRF signal was recorded after an overnight incubation at RT. LPS induces the activation of TLR4 signaling, leading to a 3-fold increase in TBK1 phosphorylation on Ser172. The expression level of the kinase remains stable which demonstrates that LPS doesn't induce any cytotoxic effect.

Analysis of TBK1 phosphorylation to monitor the activation of the cGAS-STING pathway

Mouse Kupffer cells (ImKC cell line) were plated in 96-well plates (200,000 cells/well) in complete culture medium and incubated at 37°C - 5% CO2. The day after, cells were treated for 1 hour with increasing concentrations of 23-cGAMP diluted in serum-free medium. After medium removal, cells were then lysed with 50 µL of supplemented lysis buffer #4 for 30 minutes at RT under gentle shaking, and 16 µL of lysate were transferred twice over into a low volume white microplate before adding 4 µL of the HTRF® phospho-TBK1 or total TBK1 detection antibodies. The HTRF signal was recorded after an overnight incubation at RT. Cell treatment with 23-cGAMP leads to the activation of STING which in turn triggers a 3.6-fold increase in TBK1 phosphorylation on Ser172. As expected, the expression level of the kinase remains constant.

HTRF phospho- & total TBK1 cellular assays compared to Western Blot

The human cervical cancer cell line HeLa was seeded in a T175 flask in complete culture medium, and incubated for 2 days at 37°C, 5% CO2 until 90% confluency was reached. Cells were then treated with 200 nM Calyculin A for 10 minutes and lysed with 3 mL of supplemented lysis buffer #4 for 30 minutes at RT under gentle shaking. Soluble supernatants were collected after a 10-minute centrifugation. Serial dilutions of the cell lysate were performed in the 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- or total TBK1 detection antibodies. Equal amounts of lysates were used for a side by side comparison between HTRF and Western Blot. Using HTRF® Phospho- or Total TBK1 kit, only 1,250 cells/well were sufficient to detect a significant signal while 2,500 cells were needed for minimal ECL signal detection using Western Blot. The HTRF assays are therefore 2 times more sensitive than the Western Blot technique.