HTRF Human XBP1s Detection Kit HTRF®

Hep-G2 or MCF7 cells were plated at 100,000 cells per well in a 96-well culture-treated plate in complete culture medium, and incubated overnight at 37°C, 5% CO2. These cells were treated with increasing concentrations of Thapsigargin for 4h at 37 °C, 5% CO2. They were then lysed with 50 µl of supplemented lysis buffer #1 (1X) for 30 min at RT under gentle shaking. 

After cell lysis, 16 µL of lysate were transferred into a 384-well low volume white microplate and 4 µL of the HTRF Total-XBP1 and XBP1s detection reagents were added. The HTRF signal was recorded after 18h of incubation at room temperature.

As expected, the Thapsigargin compound induced the translation of the XBP1s form with a dose-dependent increase in the XBP1s signal level. The Total XBP1 signal slightly increased with this treatment.

MCF7 cells were plated at 100,000 cells per well in a 96-well culture-treated plate in complete culture medium, and incubated overnight at 37°C, 5% CO2. Cells were co-treated with Thapsigargin (EC80: 150 nM) and increasing concentrations of 2 inhibitors (APY29 and MKC3946) for 4h at 37 °C, 5% CO2. They were then lysed with 50 µl of supplemented lysis buffer #1 (1X) for 30 min at RT under gentle shaking. 

After cell lysis, 16 µL of lysate were transferred into a 384-well low volume white microplate and 4 µL of the HTRF XBP1s detection reagents were added. The HTRF signal was recorded after 18h of incubation at room temperature.

As expected, the HTRF signal induced by Thapsigargin stimulation was well inhibited by the APY29 and MKC3946 compounds , with IC50  at 1.9 and 1.6 µM respectively, meaning that less XBP1s is translated and expressed in the presence of inihitors.

After an overnight incubation in a T175 flask , HEK 293T cells were transfected with 40 µg of human XBP1s or XBP1u plasmids and then incubated in a complete culture medium for 24h at 37°C, 5% CO2.

The cells were lysed with 3 mL of supplemented lysis buffer # 1 for 30 minutes at RT under gentle shaking, and 16 µL of lysate were transferred into a 384-well low volume white microplate before the addition of 4 µL of the HTRF XBP1s detection reagents. The HTRF signal was recorded after an overnight incubation.

No signal was detected on the HEK293T cell lysates transfected with XBP1u or non transfected, whereas a huge signal was detected in the XBP1s transfected cell lysates. This demonstrates the specificity of the assay for XBP1s protein.

Adherent human & mouse HAP1, MCF7, and Hep-G2 cells were seeded at 100,000 cells / well in a 96-well microplate. After a 24h incubation, the cells were treated with Thapsigargin for 4h at 37°C. They were then lysed with supplemented lysis buffer #1, and 16 µL of lysate were transferred into a 384-well low volume white microplate before the addition of 4 µL of the HTRF XBP1s detection reagents. The HTRF signal was recorded after an overnight incubation.

The HTRF XBP1s assay efficiently detects XBP1s protein in various cellular models expressing different levels of the protein.

MCF7 cells were cultured in a T175 flask in a complete culture medium for 24h at 37°C, 5% CO2. They were treated with 700 nM of Thapsigargin compound for 4h at 37°C, 5% CO2, 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 #1 (1x), and 16 µL of each dilution were transferred into a low volume white microplate before the addition of 4 µL of HTRF XBP1s detection reagents.

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

In these conditions, the HTRF XBP1s assay was 4 times more sensitive than the Western Blot technique.