Anti mdm2

Manufactured by BD

Sourced in United States

Anti-MDM2 is a lab equipment product that functions as an inhibitor of the MDM2 protein. MDM2 is a negative regulator of the tumor suppressor protein p53, and inhibiting MDM2 can lead to increased p53 activity. This product is intended for use in research applications.

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4 protocols using anti mdm2

Protein Extraction and Western Blot Analysis

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Protein extraction and western blot analysis were performed as previously described [39 (link)].
Cells were harvested for protein extraction 48h after transfection. Lysates from transfected cells were collected using RIPA buffer (Sigma R0278, St Louis, MO, USA). Protein lysates were separated on SDS-polyacrylamide gels, electroblotted onto polyvinylidene difluoride membrane and immunoblotted with primary antibodies, then with peroxidase-conjugated secondary antibodies. After three additional washes, the immune complex was detected by ECL detection (Thermo 34077; Rockford, IL, USA). The following antibodies were used: anti-Cyclin A2 (1540-1, Epitomics, Burlingame, CA, USA), anti-Cyclin B1(1495-1, Epitomics), anti-Cyclin D1(1677-1, Epitomics), anti-Cyclin E(4129;Cell Signaling Technology, Beverly, MA, USA), anti-Bcl-2 (1017-1, Epitomics), anti-PUMA(1652-1, Epitomics), anti-mdm2 (556353, BD PharMingen, San Diego, CA, USA), anti-Caspase-3 (1087-1, Epitomics), anti-p21(2990-1, Epitomics), anti-PARP-1 (1072-1, Epitomics), anti-Bax (1063-1, Epitomics), anti-β-actin (60008-1-Ig, Protein Tech, Chicago, IL, USA), goat anti-mouse IgG-HRP (sc-2005, Santa Cruz Biotechnology, Dallas, Texas) and goat anti-rabbit IgG-HRP (sc-2004, Santa Cruz Biotechnology).

He X., Liao J., Liu F., Yan J., Yan J., Shang H., Dou Q., Chang Y., Lin J, & Song Y. (2014). Functional repair of p53 mutation in colorectal cancer cells using trans-splicing. Oncotarget, 6(4), 2034-2045.

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Western Blot for Protein Expression Analysis

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Western blotting analysis of proteins in cell lysates was determined as previously described (Wu et al, 2014b (link)). Primary antibodies used were as follows: anti-MDM2 (Cat# 556353), anti-E-cadherin (Cat# 610405) and anti-Fibronectin (Cat# 610078) were purchased from BD (San Diego, CA, USA); anti-Slug (Cat# 9585), anti-Smad2/3 (Cat# 3102), anti-p-Smad2 (Cat# 3108), anti-p-Smad3 (Cat# 9520), anti-Smad4 (Cat# 9515) and anti-myc-tag (Cat# 2278) were from Cell Signaling Technology (Beverly, MA, USA); anti-Snail (Cat# AF3639) and anti-TGF-beta RI (Cat# AF3025) were from R&D; anti-β-Actin (Cat# sc-47778) was purchased from Santa Cruz (Santa Cruz, CA, USA).

Chen Y., Wang D.D., Wu Y.P., Su D., Zhou T.Y., Gai R.H., Fu Y.Y., Zheng L., He Q.J., Zhu H, & Yang B. (2017). MDM2 promotes epithelial–mesenchymal transition and metastasis of ovarian cancer SKOV3 cells. British Journal of Cancer, 117(8), 1192-1201.

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Western Blot Analysis of Cell Signaling

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Expression levels were performed by western-blot analysis using mouse anti-p53 and anti-β-actin (Sigma-Aldrich), anti-bax (Oncogene), anti-p21 (Beckton Dickenson), anti-Mdm2 (BD Pharmingen), anti-BCL-XL, and rat anti-HSC70 (Stressgen). Proteins were quantified by Image J software.

Mahfoudhi E., Lordier L., Marty C., Pan J., Roy A., Roy L., Rameau P., Abbes S., Debili N., Raslova H., Chang Y., Debussche L., Vainchenker W, & Plo I. (2016). P53 activation inhibits all types of hematopoietic progenitors and all stages of megakaryopoiesis. Oncotarget, 7(22), 31980-31992.

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Western Blot Analysis of Cell Lysates

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The cells from each group were collected, and lysates were generated using the KeyGEN kit (KGP2100, Nanjing, China). The protein concentration was assessed by the BCA method (Thermo Fisher Scientific, Waltham, USA). Next, equal amounts of proteins were analyzed by WB using the following antibodies: anti-MDM2 (556353, BD Pharmingen), anti-p53 (DO-1, Santa Cruz), anti-IDO1 (ab55305, Abcam), anti-p21 (ab109199, Abcam), anti-BCL2 (ab182858, Abcam), anti-BCL-XL (ab32370, Abcam), anti-PUMA (ab33906, Abcam), anti-BAX (ab32503, Abcam) and anti-β-actin (P60709, Cell Signaling Technology). The signals were detected using an ECL chemiluminescence detection system (Bio-Rad, USA). The unprocessed WB images were shown in Supplementary Fig. S4.

Sun C., Li M., Zhang L., Sun F., Chen H., Xu Y., Lan Y., Zhang L., Lu S., Zhu J., Huang J., Wang J., Hu Y., Feng Y, & Zhang Y. (2022). IDO1 plays a tumor-promoting role via MDM2-mediated suppression of the p53 pathway in diffuse large B-cell lymphoma. Cell Death & Disease, 13(6), 572.

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