XBP1s assay principle

The HTRF Human XBP1 assay quantifies the expression level of NRF2 in a cell lysate. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. This XBP1 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 presence of XBP1s 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.

XBP1 two-plate assay protocol

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 Human XBP1 HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

XBP1 one-plate assay protocol

Detection of Human XBP1 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.

Pharmacological Validation of XBP1s assay with inhibitors

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 inhibitors.

Specificity of XBP1s assay

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.

XBP1s detection in various cell lines

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.

HTRF XBP1s assay compared to Western Blot

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.

XBP1 is a key component of the Unfolded protein response (UPR) that modulates cellular survival. There are three UPR pathways involving different mediators such as ATF6, IRE1 and PERK, and leading to significant downstream effects.

Upon ER stress, and the accumulation of unfolded proteins, BiP releases its inhibitory binding of the three ER stress proteins and activates them.

When activated, IRE1 first dimerizes, then autophosphorylates which activates its mRNase activity. Through this action, IRE1 splices XBP-1u mRNA to XBP1s, enabling its translation and then its translocation to the nucleus. XBP1s then acts as a transcription factor.