HTRF Human Phospho-MDM2 (Ser166) Detection Kit, 500 Assay Points

MDM2 phospho-Ser166 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 MDM2 phospho-Ser166 HTRF detection reagents. This protocol allows the cells’ viability and confluence to be monitored.

MDM2 phospho-Ser166 one-plate assay protocol

Detection of Phospho MDM2 (Ser166) 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.

Specificity of the HTRF MDM2 phospho-Ser166 assay demonstrated by peptide competition

HCT116 cell lysates, stimulated or unstimulated with 10 µM Nutlin-3a for 24 hours, were prepared to increase phospho-MDM2 expression prior to the addition of increasing concentrations of phosphorylated and non-phosphorylated peptides, either specific or non-specific for the Ser166 residue (#9046, #9047, #9048, and #9049; SB Peptide).

8 µL of stimulated or non-stimulated cell lysate were transferred into a 384-well low-volume white microplate. Subsequently, 8 µL of phosphorylated or non-phosphorylated peptide solution were added. Finally, 4 µL of HTRF MDM2 phospho-Ser166 detection antibodies were added to assess competition between endogenous MDM2 phospho-Ser166 and the exogenous peptides. The HTRF signal was measured after an overnight incubation.

These results demonstrate the high specificity of the HTRF MDM2 phospho-Ser166 assay for the phosphorylated Ser166 epitope of MDM2, with no detectable cross-reactivity with the non-phosphorylated form or the neighboring Ser186 phosphorylation site.

Induction of MDM2 phospho-Ser166 by Nutlin-3a in A549 cells

A549 cells were seeded in 96-well plates at 25,000 cells per well and cultured for 24 hours. Cells were then treated with 10 µM Nutlin-3a or vehicle control for 16 hours, thereby indirectly increasing MDM2 expression through p53-dependent induction. Following treatment, cells were lysed with 50 µL of supplemented Lysis Buffer #4 containing 1X Blocking Reagent for 30 minutes at room temperature under gentle shaking. After lysis, 16 µL of cell lysate were transferred into a 384-well low-volume white microplate, and 4 µL of HTRF Phospho-MDM2 (Ser166) detection antibodies were added. The HTRF signal was measured after an overnight incubation at room temperature using the homogeneous, no-wash HTRF assay format for endogenous protein quantification.

As expected, Nutlin-3a treatment increased pS166-MDM2 levels. In addition, Nutlin-3a did not induce cytotoxic effects, as assessed using the ATPLite cell viability assay.

LY294002 inhibits MDM2 phospho-Ser166 levels in A549 cells

A549 cells were seeded in 96-well plates at 25,000 cells/well and cultured for 24 hours. Cells were then pre-treated with increasing concentrations of LY294002, a PI3K inhibitor, or vehicle control for 3 hours.

Subsequently, cells were treated with 10 µM Nutlin-3a for 16 hours. Following treatment, cells were lysed with 50 µL of supplemented Lysis Buffer #4 (1X) for 30 minutes at room temperature. Then, 16 µL of cell lysate were transferred into a 384-well low-volume white microplate, and 4 µL of HTRF total MDM2 detection antibodies were added. The HTRF signal was measured after an overnight incubation at room temperature.

Nutlin-3a disrupts the MDM2-p53 interaction, leading to p53 stabilization and transcriptional upregulation of MDM2. Accordingly, Nutlin-3a treatment induced an increase in total MDM2 levels. Inhibition of the PI3K/AKT pathway by LY294002 attenuated this response in a dose-dependent manner, consistent with the role of PI3K/AKT signaling in regulating MDM2 expression and activity.

Detection of MDM2 phospho-Ser166 expression across multiple human cell lines

Human U2OS, MCF-7, and A549 cells were seeded into 96-well plates at 25,000 cells/well and cultured for 24 hours. Cells were then treated with 10 µM Nutlin-3a or vehicle control for 24 hours. Nutlin-3a disrupts the MDM2-p53 interaction, leading to p53 stabilization and increased MDM2 expression, which is accompanied by elevated MDM2 phosphorylation at Ser166. Cells were lysed with 50 µL of supplemented Lysis Buffer #4 (1X) for 30 minutes at room temperature under gentle shaking.

Following lysis, 16 µL of cell lysate were transferred into a low-volume 384-well white microplate, and 4 µL of HTRF Phospho-MDM2 (Ser166) detection antibodies were added. The HTRF signal was measured after an overnight incubation at room temperature.

The HTRF MDM2 phospho-Ser166 signal increased in all tested human cell lines, demonstrating robust detection of MDM2 phosphorylated at Ser166. The observed responses in U2OS, MCF-7, and A549 cells confirm the suitability of these cellular models for MDM2 phospho-Ser166 detection. These results further support the use of the HTRF MDM2 phospho-Ser166 assay for the sensitive, robust, and reliable quantification of endogenous MDM2 phospho-Ser166 across multiple human cell lines.

MDM2 phospho-Ser166 signaling pathway

MDM2 is an oncogenic protein encoded by the MDM2 gene and functions as an E3 ubiquitin ligase that negatively regulates the tumor suppressor p53. MDM2 binds directly to the transactivation domain of p53, inhibiting its transcriptional activity, and promotes its ubiquitination and proteasomal degradation, thereby maintaining low p53 levels under normal conditions. The MDM2-p53 axis forms a negative feedback loop in which p53 induces MDM2 transcription, while MDM2 suppresses p53 activity.

In addition to its p53-dependent role, MDM2 participates in several p53-independent cellular processes, including cell cycle progression and DNA repair. MDM2 activity is tightly regulated by multiple signaling pathways. In response to growth factor stimulation, the PI3K/AKT pathway phosphorylates MDM2 at Ser166, promoting its nuclear localization and enhancing its ability to inhibit p53. Conversely, in response to DNA damage, the ATM and ATR signaling pathways trigger phosphorylation events on MDM2, p53, and downstream effector proteins, resulting in p53 stabilization and activation of the DNA damage response. Together, these pathways integrate proliferative and stress signals and play a central role in regulating cell fate through the MDM2-p53 axis.

MDM2 is frequently amplified or overexpressed in a wide range of human cancers, resulting in the suppression of p53 activity even in tumors harboring wild-type TP53. This dysregulation enables cancer cells to evade cell cycle arrest and apoptosis. Consequently, MDM2 has emerged as an attractive therapeutic target, and numerous inhibitors of the MDM2-p53 interaction are currently being developed to restore p53 function and promote antitumor activity.