Figure 1: The crystal structure of MDM2 (3-109) and P73 TAD (10-25).

Introduction to MDM2

Protein Function

• MDM2 is an E3 ubiquitin-protein ligase involved in various proteins' ubiquitination and subsequent degradation. One of its primary functions is mediating the ubiquitination of p53/TP53, leading to its proteasomal degradation.
• Inhibits p53/TP53- and p73/TP73-mediated cell cycle arrest and apoptosis by binding its transcriptional activation domain.

Protein Expression

• Isoforms Mdm2-A, B, C, D, E, F, and G are observed in various cancers but absent in normal tissues.

Protein Localization

• Expressed predominantly in the nucleoplasm. Interaction with ARF(P14) results in the localization of both proteins to the nucleolus. There are also reports of its localization in the cytoplasm.

Figure 2: MDM2 ICC Image, Immunocytochemistry/Immunofluorescence - Anti-MDM2 antibody [EPR22256-98] (ab259265). Green: MDM2; Red: alpha Tubulin; Blue: DAPI. The confocal image shows enhanced nuclear staining in the HepG2 cell line after 24 hours of treatment with Nutlin-3a (10 µM).

Isoforms & Post-Translational Modifications

• Human (Q00987): Isoform 1, 8-11, 48-55 kDa; Isoforms 2-5, 24-35 kDa; Isoforms 6-7, 11-14 kDa (predicted)
• Mouse (P23804): Isoforms 1-2, 49-54 kDa (predicted)
• Multiple phosphorylation sites.

WB Experiment Tips

Precautions

• MDM2 exhibits significant expression specificity, being more easily detectable in cancer tissues but showing low or no expression in normal tissues, which might result in undetectable signals.
  Some cell lines require induction stimuli to detect MDM2 protein. Please choose appropriate induction conditions based on literature or product instructions.
• If you are unsure whether your sample expresses the target protein, we strongly recommend using a positive control in your experiment.
• MDM2 is localized in the nucleus. We recommend using sonication to lyse the cells to obtain more target protein. If the signal is still weak in whole cell lysates, try extracting the nuclear fraction to enhance detection.
• The predicted molecular weight of MDM2 is 55 kDa. However, due to the presence of various isoforms, post-translational modifications, and cleavage by p53 activation, the observed molecular weights can be 90 kDa or 60 kDa. Therefore, multiple bands might be observed. We advise not cutting the membrane.

Positive Control

• 2.4G2 treated with 10 µM Nutlin-3a for 24 hours whole cell lysate.

Example Results

Figure 3: Anti-MDM2 antibody [EPR22256-98] (ab259265).

Lane 1: 20 µg Untreated RAW264.7 whole cell lysate
Lane 2: 20 µg RAW264.7 treated with 10 µM Nutlin-3a for 24 hours whole cell lysate
Lane 3: 20 µg 2.4G2 whole cell lysate
Lane 4: 20 µg 2.4G2 treated with 10 µM Nutlin-3a for 24 hours whole cell lysate

Predicted band size: 55 kDa
Observed band sizes: 60, 90 kDa

Key control points

In the experiment, in addition to paying attention to routine issues, special attention should be paid to the following key control points:

Sample preparation:

1. Add a protease inhibitor cocktail to prevent degradation of target proteins.
2. Sonicate cell samples to release your target protein into solution and obtain a higher yield.
3. Keep samples on ice throughout the entire sample preparation process.
4. Determine the protein concentration of the samples using Bradford analysis, Lowry analysis, or BCA analysis.
5. We recommend using positive control.

Electrophoresis:

1. Load at least 20 μg of total protein from cell lysate or tissue homogenate.

Transfer:

1. We recommend staining the membrane with Ponceau S after the transfer to confirm its success (if using fluorescent detection, ensure thorough washing after Ponceau S staining).

Antibody incubation:

1. Select a suitable antibody working concentration according to the product datasheet.

References

1. Leonard Girnita, Ada Girnita, Olle Larsson. Mdm2-dependent ubiquitination and degradation of the insulin-like growth factor 1 receptor. Proc Natl Acad Sci U S A. 2003 Jul 8;100(14):8247-52. doi: 10.1073/pnas.1431613100. Epub 2003 Jun 23.
2. Muyang Li, Christopher L Brooks, Ning Kon, Wei Gu. A dynamic role of HAUSP in the p53-Mdm2 pathway. Mol Cell. 2004 Mar 26;13(6):879-86. doi: 10.1016/s1097-2765(04)00157-1.
3. Naoe Taira, Hiroyuki Yamamoto, Tomoko Yamaguchi, Yoshio Miki, Kiyotsugu Yoshida. ATM augments nuclear stabilization of DYRK2 by inhibiting MDM2 in the apoptotic response to DNA damage. J Biol Chem. 2010 Feb 12;285(7):4909-19. doi: 10.1074/jbc.M109.042341. Epub 2009 Dec 4.
4. Rana Elkholi, Ioana Abraham-Enachescu, Andrew P Trotta, etc. MDM2 Integrates Cellular Respiration and Apoptotic Signaling through NDUFS1 and the Mitochondrial Network. Mol Cell. 2019 May 2;74(3):452-465.e7. doi: 10.1016/j.molcel.2019.02.012. Epub 2019 Mar 14.
5. Lily I Huschtscha, Jonathan D Moore, Jane R Noble, Hamish G Campbell, Janice A Royds, Antony W Braithwaite, Roger R Reddel. Normal human mammary epithelial cells proliferate rapidly in the presence of elevated levels of the tumor suppressors p53 and p21(WAF1/CIP1). J Cell Sci. 2009 Aug 15;122(Pt 16):2989-95. doi: 10.1242/jcs.044107. Epub 2009 Jul 28.
6. R Pochampally, B Fodera, L Chen, W Lu, J Chen. Activation of an MDM2-specific caspase by p53 in the absence of apoptosis. J Biol Chem. 1999 May 21;274(21):15271-7. doi: 10.1074/jbc.274.21.15271.
7. Kaleigh Canfield, Wendy Wells, Joseph Geradts, William B Kinlaw, Chao Cheng, Manabu Kurokawa. Inverse association between MDM2 and HUWE1 protein expression levels in human breast cancer and liposarcoma. Int J Clin Exp Pathol. 2016;9(6):6342-6349. Epub 2016 Jun 15.