Total GSPT1 assay principle

The HTRF Total GSPT1 assay quantifies the expression level of GSPT1 in a cell lysate. Unlike Western Blot, the assay is entirely plate-based, and does not require gels, electrophoresis, or transfer. The Total GSPT1 assay uses two labeled antibodies, one coupled to a donor fluorophore, the other to an acceptor. Both antibodies are highly specific for a distinct epitope on the protein. In presence of GSPT1 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.

Total GSPT1 two-plate assay protocol

The 2-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 the Total GSPT1 HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

Human Total GSPT1 one-plate assay protocol

Detection of Total GSPT1 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.

HTRF Total GSPT1 modulation using molecular glues on Human NSCLC cells NCI-H358

The Human NSCLC cells NCI-H358 were seeded in 96-well culture plates at a density of 30,000 cells/well for 24 hours at 37°C, 5% CO2. Cells were treated with increasing concentrations of the molecular glue MRT-2359 for 4h or 24h. 1µM of the proteasome inhibitor epoxomicin was added or not 1h prior to the addition of MRT-2359.

After treatment, the cell culture medium was removed and 50 µl of supplemented Lysis Buffer#4 (1X) were dispensed into each well. Then 16 µL of lysates were transferred into a 384-well low volume white microplate, and 4 µL of the HTRF GSPT1 detection antibodies were added. An additional 4 µL of lysates (supplemented with 12 µL diluent #8) were also transferred into the microplate to check the alpha-tubulin level with the Alpha-tubulin Housekeeping Cellular Kit (64ATUBPEG). HTRF signals were recorded after an overnight incubation.

The MRT-2359 molecular glue induced a dose-dependent decrease in the GSPT1 protein with DC50* of 153 nM and 93 nM after 4h and 24h respectively. Furthermore, MRT-2359 induced GSPT1 degradation was prevented with epoxomicin, clearly showing the involvement of the ubiquitin proteasome system in MRT-2359 mediated GSPT1 degradation.

DC50 corresponds to the concentration of the degrader at which 50% of the targeted protein is degraded.

Specificity of HTRF Total GSPT1 assay using siRNA

HEK293 cells were seeded in 96-well culture plates at a density of 10,000 cells/well and incubated for 24 hours at 37°C, 5% CO2. The cells were then transfected with siRNAs specific for GSPT1 or GSPT2 as well as with a negative control siRNA. After a 48h incubation, the cells were lyzed and 16 µL of lysates were transferred into a 384-well low volume white microplate before the addition of 4 µL of the HTRF Total GSPT1 detection antibodies. HTRF signals were recorded after an overnight incubation.

The GSPT1 siRNA led to a 70% signal decrease compared to the cells transfected with the negative siRNA. No signal decrease was observed in cells transfected with GSPT2 siRNA. In the same experimental conditions, no cytotoxic effects were observed, as evidenced by ATP levels measured with ATPliteTM assay (data not shown). These results demontrate the detection specificity of the HTRF GSPT1 Total kit.

Comparison between HTRF and Western Blot sensitivity for Total GSPT1

HEK293 cells were cultured in a T175 flask in complete medium at 37°C, 5% CO2, until confluency was reached.

After medium removal, the cells were lysed with 3 mL of supplemented lysis buffer #4 (1x) for 30 min at RT under gentle shaking.

Serial dilutions of the cell lysate were performed using supplemented lysis buffer #4 (1x), and 16µL of pure sample and of each dilution were transferred into a 384-well small volume microplate before the addition of 4µL of HTRF Total GSPT1 detection antibodies. HTRF signals were recorded after an overnight incubation.

Equal amounts of lysates were loaded into a gel for a side by side comparison between HTRF and Western Blot.

In these conditions, the HTRF Total GSPT1 assay is 256-fold more sensitive than the Western Blot technique.

GSPT1 Signaling Pathway

Translation is initiated when a ribosome is recruited to the 5 end of mRNA. The eIF4E-binding proteins, such as 4EBP1, block translation by binding to eIF4E and preventing recruitment of the translation machinery. 4EBP1 hyperphosphorylation by mTOR abrogates eIF4E-binding activity and therefore promotes translation.

GSPT1, also known as eukaryotic release factor 3a (eRF3a), function as a small GTPase to mediate stop codon recognition.

Through its C-terminus, GSPT1 interact with eRF1, another eukaryotic release factor, to form part of the translation termination complex forcing the proteins to dissociate when a stop codon enters the A site of the ribosome.