HTRF Human Phospho-FOXM1 (Thr600) Detection Kit, 500 assay points

Phospho-FOXM1 (Thr600) assay principle

The Phospho-FOXM1 (Thr600) assay measures FOXM1 when phosphorylated at Thr600. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The assay uses 2 antibodies, one labeled with a donor fluorophore and the other with an acceptor. The first antibody was selected for its specific binding to the phosphorylated motif on the protein, and the second for its ability to recognize the protein independently of its phosphorylation state. Protein phosphorylation allows an immune-complex formation involving both labeled antibodies. This brings the donor fluorophore into close proximity to the acceptor, thereby generating a FRET signal. Its intensity is directly proportional to the concentration of phosphorylated protein present in the sample and provides a means of assessing the protein's phosphorylation state under a no-wash assay format.

Phospho-FOXM1 (Thr600) 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 Phospho-FOXM1 (Thr600) HTRF detection reagents. This protocol allows the cells' viability and confluence to be monitored.

Phospho-FOXM1 (Thr600) one-plate assay protocol

Detection of Phosphorylated FOXM1 (Thr600) 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 allows miniaturization while maintaining robust HTRF quality.

Phospho-FOXM1 (Thr600) modulation using Nocodazole

HeLa cells were seeded in a 96-well culture plate (100,000 cells/well) in complete culture medium and incubated for 24 hours at 37°C with 5% CO₂. Cells were then treated for 20 hours with increasing concentrations of Nocodazole.

After cell lysis, 16 µL of lysates were transferred into a 384-well low volume white microplate, and 4 µL of the HTRF Total FOXM1 or Phospho-FOXM1 (Thr600) detection antibodies were added. The HTRF signal was recorded after 4 hours of incubation.

As expected, Nocodazole triggered a dose-dependent increase in total and phosphorylated FOXM1 at Thr600.

Pharmacological modulation of phospho-FOXM1 (T600) using Palbociclib

HCT-116 cells were seeded in a 96-well culture plate (100,000 cells/well) in complete culture medium and incubated for 24 hours at 37°C with 5% CO₂. Cells were then co-treated for 20 hours with increasing concentrations of Palbociclib and 0.5µM of Nocodazole.

After cell lysis, 16 µL of lysates were transferred into a 384-well low volume white microplate, and 4 µL of the Phospho-FOXM1 (Thr600) or HTRF Total FOXM1 detection antibodies were added. The HTRF signal was recorded after 4 hours of incubation.

As expected, Nocodazole with Palbociclib (CDK4/6 inhibitor) treatment induced a decrease in both total and phospho- FOXM1 (Thr600) to the basal level.

Validation of the specificity of phospho-FOXM1 (Thr600) using HAP1 cells knocked out for FOXM1

The phospho FOXM1 protein levels were assessed with the HTRF Human phospho-FOXM1 (T600) kit in HAP1 cells (WT) and a HAP1 cell line Knocked-Out for FOXM1. The cell density was optimized beforehand to ensure HTRF detection within the dynamic range of the kit (data not shown).

The cells were cultured in a 96-well plate (100,000 cells/well) for 24 hours at 37°C, 5% CO₂, followed by Nocodazole treatment at 0.5µM for 20 hours. The cells were then lysed with 50 µL of supplemented lysis buffer #1 (1X) for 30 minutes at RT under gentle shaking, and 16 µL of cell lysate were transferred into a low volume white microplate, followed by 4 µL of premixed phospho FOXM1 detection reagents. The HTRF signal was recorded after 4 hours of incubation at RT.

In HAP1 KO FOXM1 cells, the HTRF signal decreased by 91%, indicating a significant FOXM1 gene silencing, whereas the phospho- FOXM1 level was well detected in the other cell line, as expected. This demonstrates that the HTRF Phospho-FOXM1 (T600) assay is specific for Phospho-FOXM1 detection.

Catalog cell line references (Horizon Discovery): HAP1 Wt #C631; HAP1 KO FOXM1 #HZGHC000835c006

Assessment of phospho-FOXM1 (Thr600) levels in various cell lines

Adherent (A431, HeLa, HAP1, & HCT-116) and suspension (RAJI) human cells were seeded at 100,000 cells/well in a 96-well microplate. After 24h of incubation, the cells were treated with 0.5µM Nocodazole for 20 hours. The cells were then lysed for 30 minutes with supplemented lysis buffer #1, following the protocol for adherent or suspended cells at RT under gentle shaking.

16 µL of lysate were transferred into a 384-well low volume white microplate before the addition of 4 µL of the HTRF phospho-FOXM1 (Thr600) detection reagents. The HTRF signal was recorded after 4 hours of incubation.

The HTRF phospho-FOXM1 (Thr600) assay efficiently detected phospho-FOXM1 in various cellular models expressing different levels of the protein.

HTRF FOXM1 Phospho-T600 assay compared to Western Blot

HeLa cells were grown in a T175 flask in complete culture medium at 37°C, 5% CO₂, until 80% confluence. After a 24h incubation, the cells were stimulated with 0.5µM of Nocodazole for 20 hours. They were 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 FOXM1 (T600) detection reagents. Equal amounts of lysates were used for a side-by-side comparison between HTRF and Western Blot.

In these conditions, the HTRF Phospho FOXM1 (T600) assay was 4 times more sensitive than the Western Blot technique.

FOXM1 signaling pathway

FOXM1 is a regulatory factor involved in the G1/S and G2/M phases of the cell cycle. Its activity is controlled by multisite phosphorylation and ubiquitination throughout the cell cycle. The transcriptional activity of FOXM1 is tightly regulated by phosphorylation at multiple sites by various kinases and phosphatases, as well as by proteasomal degradation. During the S phase, FOXM1 is phosphorylated by cyclin E/A–CDK2 complexes, followed by hyperphosphorylation in G2 and M phases by cyclin B–CDK1. Several genes and transcription factors, including p53, WDR5, and HIF‑1α, regulate FOXM1 expression, contributing to increased cell invasion, epithelial–mesenchymal transition (EMT), inhibition of apoptosis, and enhanced cell survival.