Anti mtdh

Manufactured by Cell Signaling Technology

Sourced in United States

Anti-MTDH is a primary antibody that recognizes the Metadherin (MTDH) protein. MTDH is a transmembrane protein involved in various cellular processes. This antibody can be used to detect and study the MTDH protein in various experimental applications.

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4 protocols using anti mtdh

Western Blot Analysis of MTDH Protein

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Protein extracts were separated by electrophoresis in a sodium dodecyl sulfate-polyacrylamide gel (Invitrogen, Camarillo, CA, USA) and transferred onto polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA) for immunoblotting. The membranes were hybridized with a primary antibody at 4 °C overnight followed by incubation with a secondary antibody for 1 h at room temperature. β-actin (Santa Cruz, Shanghai, China) was used as the loading control. Signals were visualized using a chemiluminescent detection system (Thermo Scientific, Rockford, IL, USA) and exposure to the film. The antibodies used for western blotting is anti-MTDH (Cell Signaling, Shanghai, China).

Zhao J., Zhou K., Ma L, & Zhang H. (2020). MicroRNA-145 overexpression inhibits neuroblastoma tumorigenesis in vitro and in vivo. Bioengineered, 11(1), 219-228.

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Immunofluorescence Staining of Cell Markers

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Cells were seeded on glass cover slips and fixed with 4% formaldehyde in PBS. Immunofluorescence staining was done with anti-MTDH, anti-PTEN, anti-p-AKT and anti-p65 (Cell Signaling Technology, MA, USA) antibodies. Slips were incubated with indicated primary antibodies, followed by incubation with fluorescein isothiocyanate-conjugated goat anti-mouse secondary antibody or Texas Red-conjugated goat-anti-rabbit secondary antibody (Abcam, Cambridge, MA, USA). Then slips were counterstained with 4’,6-diamidino-2- phenylindole dihydrochloride (DAPI) solution (Sigma, St Louis, MO, USA). Slips were observed and imaged under fluorescence microscope.

Du C., Yi X., Liu W., Han T., Liu Z., Ding Z., Zheng Z., Piao Y., Yuan J., Han Y., Xie M, & Xie X. (2014). MTDH mediates trastuzumab resistance in HER2 positive breast cancer by decreasing PTEN expression through an NFκB-dependent pathway. BMC Cancer, 14, 869.

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Western Blot Analysis of MTDH, Bax, and Bcl-2

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Protein was isolated from MCF-7 cells using RIPA lysis buffer (Thermo Fisher Scientific), followed by quantification using a BCA kit (Qiagen). Protein was separated onto sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electroblotted onto polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA) according to standard methods. Membranes were then incubated with specific primary antibody anti-MTDH (Sigma), anti-Bax (Cell Signaling Technology [CST], Danvers, MA, USA), anti-Bcl-2 (CST), and anti-β-actin (CST) at 4°C overnight and related secondary antibodies at room temperature for 40 minutes. Bio-Rad Quantity One software (Bio-Rad) was used to analyze protein expression with the enhanced chemiluminescence system.

Tian W., Hao S., Gao B., Jiang Y., Zhang X., Zhang S., Guo L., Zhao J., Zhang G., Chen Y., Li Z, & Luo D. (2018). Lobaplatin inhibits breast cancer progression, cell proliferation while it induces cell apoptosis by downregulating MTDH expression. Drug Design, Development and Therapy, 12, 3563-3571.

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Immunofluorescence Staining of MTDH and γH2AX

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Hec50 cells were seeded on coverslips then fixed with 2% paraformaldehyde for 20 min. Coverslips were rinsed 3 times with 1ml PBS and incubated with 80% ice-cold methanol for 1 h, followed by permeabilization for 25–30 min with 0.2% Triton X-100. Cells were blocked with 3% BSA then incubated with specific antibodies at 4 °C overnight. Anti-MTDH (1:100, #14065), anti-phospho-histone H2AX (Ser139) (1:400, #9718) were from Cell Signaling Technology (Danvers, MA). Then, cells were incubated with Alexa Fluor 546-conjugated anti-rabbit secondary antibody (1:200, Cell Signaling Technology) at room temperature for 2 h; nuclei were stained using mounting solution with DAPI (Vector Laboratories). Visualization was performed on a Zeiss 710 confocal microscope.

Bi J., Areecheewakul S., Li Y., Yang S., Zhang Y., Ebeid K., Li L., Thiel K.W., Zhang J., Dai D., Salem A.K., Leslie K.K, & Meng X. (2019). MTDH/AEG-1 downregulation using pristimerin-loaded nanoparticles inhibits Fanconi anemia proteins and increases sensitivity to platinum-based chemotherapy. Gynecologic oncology, 155(2), 349-358.

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