Vimentin Overexpression Lysate

HEK293T cells in 10-cm dishes were transiently transfected with a non-lipid polymer transfection reagent specially designed and manufactured for large volume DNA transfection. Transfected cells were cultured for 48hrs before collection. The cells were lysed in modified RIPA buffer (25mM Tris-HCl pH7.6, 150mM NaCl, 1% NP-40, 1mM EDTA, 1xProteinase inhibitor cocktail mix, 1mM PMSF and 1mM Na3VO4, and then centrifuged to clarify the lysate. Protein concentration was measured by BCA protein assay kit.

Vimentin is a type III intermediate filament (IF) protein, encoded by the VIM gene, that plays an essential role in cytoskeletal function and is largely expressed by mesenchymal cell such as endothelial cells, fibroblasts, and immune cells (1). Human vimentin monomer protein is 466 amino acids (aa) in length with a theoretical molecular weight of ~54 kDa (2). Vimentin protein contains a central rod domain composed of four alpha-helices which is flanked by an N-terminal head domain and C-terminal tail domain (1,3). Vimentin monomers dimerize to form more complex structures resulting in the assembly of eight tetramers, referred to as unit-length filaments, which then assemble into full-length intermediate filaments that are 10-12 nm wide (1,3,4).

Activated macrophages have been shown to secrete phosphorylated vimentin which can be stimulated by a variety of pathophysiological factors including oxidized low-density lipoproteins and TNF-alpha or inhibited by IL-10 (1). The vimentin protein is often expressed at the cell surface playing a role in cell-cell interactions, tissue damage and repair, immune response, and pathogen recognition (1). Vimentin functions in many cytoskeletal processes including cell migration, which is highlighted by its upregulation during epithelial-to-mesenchymal transition (EMT) (4,5). Vimentin is a commonly used marker for EMT and is expressed by many tumor types (5). For example, high metastasis of oral squamous cell carcinomas also showed high vimentin positive expression in immunohistochemical staining analysis (5). A number of vimentin targeting compounds are in cancer-related clinical trials, however, given the multifunctional role of vimentin, the effect of inhibition on non-malignant cells needs to be thoroughly examined (5).

References

1. Ramos, I., Stamatakis, K., Oeste, C. L., & Perez-Sala, D. (2020). Vimentin as a Multifaceted Player and Potential Therapeutic Target in Viral Infections. International Journal of Molecular Sciences. https://doi.org/10.3390/ijms21134675

2. Uniprot (P08670)

3. Morrow, C. S., & Moore, D. L. (2020). Vimentin's side gig: Regulating cellular proteostasis in mammalian systems. Cytoskeleton (Hoboken, N.J.). https://doi.org/10.1002/cm.21645

4. van Bodegraven, E. J., & Etienne-Manneville, S. (2020). Intermediate filaments against actomyosin: the david and goliath of cell migration. Current Opinion in Cell Biology. https://doi.org/10.1016/j.ceb.2020.05.006

5. Strouhalova, K., Prechova, M., Gandalovicova, A., Brabek, J., Gregor, M., & Rosel, D. (2020). Vimentin Intermediate Filaments as Potential Target for Cancer Treatment. Cancers. https://doi.org/10.3390/cancers12010184

This product is for research use only and is not approved for use in humans or in clinical diagnosis. Lysates are guaranteed for 6 months from date of receipt.

Array NBP2-04249

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