Vimentin

Manufactured by Santa Cruz Biotechnology

Sourced in United States, United Kingdom, China, Germany, Canada

Vimentin is a cytoskeletal protein that is widely expressed in various cell types. It is a type III intermediate filament protein that plays a role in maintaining cell structure, cell signaling, and cell migration. Vimentin is commonly used as a marker for mesenchymal cells and is often used in research applications to study cell biology and developmental processes.

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Lab products found in correlation

473 protocols using vimentin

EMT Biomarker Immunohistochemistry

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Eleven paired formalin-fixed tissue samples from our previous study (ALK1, ALK4, ALK5, ALK6, ALK14, ALK16, ALK17, ALK20, ALK22, ALK25, and ALK26) were used for IHC staining of two EMT biomarkers: E-cadherin (using mouse monoclonal E-cadherin 1:100, clone 36/E-cadherin; BD Biosciences, Breda, The Netherlands) and Vimentin (using mouse monoclonal Vimentin 1:100, clone sc-6260; Santa Cruz Biotechnology; Bioconnect, Huissen, The Netherlands).

Wei J., van der Wekken A.J., Saber A., Terpstra M.M., Schuuring E., Timens W., Hiltermann T.J., Groen H.J., van den Berg A, & Kok K. (2018). Mutations in EMT-Related Genes in ALK Positive Crizotinib Resistant Non-Small Cell Lung Cancers. Cancers, 10(1), 10.

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Multiplex Protein Expression Analysis

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Antibodies used for western blotting include GAD1/2, EphA2, PDGFR (Cellsignal®), E-cadherin (BD Biosciences), Vimentin (SantaCruz), OVOL2, CBFA2T2, SOX2, ASCL1, POU5F1, GAPDH, SOX2, FYN, beta-actin (Sigma®). Fluorophore-conjugated primary antibodies were used for flow cytometry – CD56 BV605, CD151 PE, CD24 BUV395 (BD Biosciences), CD44 Pacific blue, E-cadherin Pacific blue (Cellsignal®), CADM1 A647 (MBL), EPHA2 A488 (R&D Systems), CD133 PE-Cy7 (Biolegend), Vimentin (Santacruz);

Udyavar A.R., Wooten D.J., Hoeksema M., Bansal M., Califano A., Estrada L., Schnell S., Irish J.M., Massion P.P, & Quaranta V. (2016). A transcription factor network model explains heterogeneity and reveals a novel hybrid phenotype in Small-Cell Lung Cancer. Cancer research, 77(5), 1063-1074.

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Immunofluorescence Staining Protocol

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For immunofluorescence staining, once cells reached 3 h of WFA incubation, they were washed in warm PBS, then fixed in 37 °C 4% PFA for 10 min and finally washed 3 times in PBS. Next, they were permeabilised with 0.25% Triton-x for 5 min and washed three times. Following this, they were blocked in 10% BSA for 1 h at room temperature, incubated with primary antibodies Keratin-14 (Santa Cruz, CA, USA, sc-58724), α -tubulin (, Cambridge, UK, ab4074), at 1:200, or Vimentin (Santa-Cruz, CA, USA, sc-32322) (1:50 to account for the increased number of cells in sheets) in 1% BSA overnight at 4 °C and washed 3 times in PBS (we have successfully used this Vimentin antibody in other cell types to image filaments). Next, they were incubated with Alexa Fluor secondary antibodies (488/555), DAPI (Sigma) and phalloidin 633 (Santa Cruz, CA, USA) (all 1:1000) for 1 h at room temperature. After washing in PBS three times, slides were mounted using Prolong Glass. The mounting media was allowed to cure for at least 24 h before use.

Keeling M.C, & Gavara N. (2020). Withaferin-A Can Be Used to Modulate the Keratin Network of Intermediate Filaments in Human Epidermal Keratinocytes. International Journal of Molecular Sciences, 21(12), 4450.

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Western Blot Analysis of Protein Expression

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Proteins (30 μg) were resolved on a 4–12% Bis–Tris gradient SDS–PAGE under reducing conditions and transferred onto nitrocellulose membrane. The primary antibodies were RANKL, E-cadherin, vimentin, OPG, c-Met (Santa Cruz Biotechnology, Inc.), p-c-Met (Tyr-1230/34/35; Invitrogen), RANK (Amgen, Thousand Oaks, CA, USA), and N-cadherin (BD Transduction Laboratories, San Jose, CA, USA). AR (441), Chr-A (H-300), synaptophysin (SYP; D4), CD44 (DF1485), Sox-2 (Y-17), Nanog (5A10), LIN-28 (H-44) (Santa Cruz Biotechnology, Inc.), FOXA2 (D56D6; Cell Signaling Technology, Danvers, MA, USA), and PROM1 (CD133; Abnova, Taipei City, Taiwan) antibodies were used to detect neuroendocrine and stem cell differentiation. c-Myc (D84C12XP), Lamin A/C (Cell Signaling Technology), and Max (sc-197X) antibodies (Santa Cruz Biotechnology, Inc.) were used for nuclear protein detection.

Chu G.C., Zhau H.E., Wang R., Rogatko A., Feng X., Zayzafoon M., Liu Y., Farach-Carson M.C., You S., Kim J., Freeman M.R, & Chung L.W. (2014). RANK- and c-Met-mediated signal network promotes prostate cancer metastatic colonization. Endocrine-Related Cancer, 21(2), 311-326.

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LINC01488 Regulates Tumor Formation and Metastasis

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In model I, nude mice were subcutaneously injected with LINC01488-depleted or -overexpressing SK-Hep1 cells (1 × 10⁶) to assess the effects on tumor formation ability. Tumor volumes (mm³) were measured using the formula (W² × L)/2 (W, smallest diameter; L, longest diameter). In model II, severe combined immunodeficient (SCID) mice were employed to determine the invasive capability of LINC01488-depleted and -overexpressing SK-Hep1 cells following intravenous injection (2 × 10⁶ cells). All animals were sacrificed on week 8 after tumor inoculation, and livers and lungs removed. Formaldehyde-fixed and paraffin-embedded tissues from lungs of SCID mice were examined by immunohistochemistry (IHC) assay using vimentin, cyclin E (Santa Cruz) and cyclin D (Abcam) antibody. Positive staining, indicating tumor cells, appeared as a brown color showing vimentin, cyclin E and cyclin D immunoreactivity. Animal experiments were performed according to the guidelines of United States National Institutes of Health and the Chang Gang Institutional Animal Care and Use Committee Guide for the Care and Use of Laboratory Animals.

Lin S.L., Lin Y.H., Chi H.C., Lin T.K., Chen W.J., Yeh C.T, & Lin K.H. (2020). A Novel Long Non-Coding RNA-01488 Suppressed Metastasis and Tumorigenesis by Inducing miRNAs That Reduce Vimentin Expression and Ubiquitination of Cyclin E. Cells, 9(6), 1504.

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Western Blot Analysis of EMT Markers

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RIPA cell lysis buffer (Beyotime Institute of Biotechnology) was used to obtain cell lysates. Proteins were quantified using bicinchoninic acid protein assay kit (Thermo Fisher Scientific, Inc.) then 20 µg protein were loaded per lane and separated via SDS-PAGE on a 10% gel. Separated proteins were then transferred to polyvinylidene difluoride membranes. Following blocking with 5% fat-free milk in PBS for 2 h at room temperature, the membranes were incubated with primary antibodies against GAPDH (cat. no. MB001; 1:2,000; Bioworld Technology, Inc.), NRP-1 (cat. no. ab81321; 1:1,000; Abcam), N-cadherin (cat. no. sc-59987; 1:1,000; Santa Cruz Technology, Inc.), vimentin (cat. no. BS1855; 1:1,000; Bioworld Technology, Inc.), E-cadherin (cat. no. sc-71007; 1:1,000; Santa Cruz Technology, Inc.) and β-catenin (cat. no. ab32572; 1:1,000; Abcam) at 4°C overnight. Membranes were then incubated with horseradish peroxidase-conjugated goat anti-mouse (cat. no. BS12478) or anti-rabbit (cat. no. BS13278; both 1:5,000; Bioworld Technology, Inc.) secondary antibodies for 1 h at room temperature. Following washing, the proteins of interest were visualized by enhanced chemiluminescence Plus Western Blotting Substrate (Thermo Fisher Scientific, Inc.) and ChemiDoc Gel Imaging System (Bio-Rad Laboratories, Inc.).

Hang C., Yan H.S., Gong C., Gao H., Mao Q.H, & Zhu J.X. (2019). MicroRNA-9 inhibits gastric cancer cell proliferation and migration by targeting neuropilin-1. Experimental and Therapeutic Medicine, 18(4), 2524-2530.

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Western Blot Assay Protocol

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Western bot assays were performed following previously described method (24). Cell lysates were made in RIPA buffer containing 20 mM Tris-HCL, 150 mM NaCl, 10% sodium deoxycholate, SDS (0.025%) supplemented with 1X protease inhibitor cocktail (Roche). The antibodies used in the study were DDB2 (#5416), N-Cadherin (#14215), Cell Signaling technology, E-cadherin (SC-7870), Vimentin (SC-6260), and HRP–conjugated secondary antibodies were purchased from Santa Cruz Biotechnology.

Bommi P.V., Ravindran S., Raychaudhuri P, & Bagchi S. (2018). DDB2 regulates Epithelial-to-Mesenchymal Transition (EMT) in Oral/Head and Neck Squamous Cell Carcinoma. Oncotarget, 9(78), 34708-34718.

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Gemcitabine-Resistant Pancreatic Cancer Cell Lines

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The human pancreatic AsPC-1 and PANC-1 cells were cultured in RPMI 1600 (Invitrogen, Carlsbad, CA, USA) and DMEM (Gibco, Gaithersburg, MD, USA), respectively, supplemented with 10% fetal bovine serum (FBS) in 5% CO₂ at 37^oC. AsPC-1 and PANC-1 cells were exposed to escalating concentrations of gemcitabine for 6 months to create gemcitabine-resistant cell lines AsPC-1 GR and PANC-1 GR. MTT [3-(4,5-dimethythiazol- 2-yl)-2,5-diphenyl tetrazolium bromide] was purchased from Sigma (St. Louis, Mo, USA). Antibodies against Vimentin, E-cadherin, Snail, Slug, ZEB1, ZEB2, Fbw7, β-actin and the secondary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Transwell inserts and Matrigel were purchased from BD Biosciences.

Ma J., Fang B., Zeng F., Ma C., Pang H., Cheng L., Shi Y., Wang H., Yin B., Xia J, & Wang Z. (2015). Down-regulation of miR-223 reverses epithelial-mesenchymal transition in gemcitabine-resistant pancreatic cancer cells. Oncotarget, 6(3), 1740-1749.

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Western Blot Analysis of Protein Expression

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Cells pellets were lysed in RIPA buffer (Sigma-Aldrich). Supernatant containing 20 µg of proteins was resolved in SDS-polyacrylamide gel and electroblotted onto PVDF membranes (Millipore, Billerica, MA) using a semidry transfer unit (ATTO, Tokyo, Japan). Immunoblotting was performed with anti-VEGF, MMP-2, Vimentin (Santa Cruz), N-Cadherin, uPA & hnRNP-K (Abcam), phospho-p38MAPK, E-Cadherin (Cell Signaling), HSP27 (StressMarq Biosciences Inc.) and β-actin (Abcam, Cambridge) antibodies. The membranes probed with the first antibodies were excessively washed with TBS-T (Tris-buffered saline-Tween 20) and incubated with secondary horseradish peroxidase (HRP)-conjugated goat anti-mouse or anti-rabbit (Santa Cruz) antibodies. Protein bands were detected using ECL prime substrate (GE Healthcare, CA). Densitometric quantitation of three independent immunoblotting experiments was performed with the Image J software (NIH, Bethesda, MD). Expression level of each of the proteins in control and treated cells was calculated with respect to the β-actin (loading control). All experiments were performed in triplicate.

Chaudhary A., Kalra R.S., Malik V., Katiyar S.P., Sundar D., Kaul S.C, & Wadhwa R. (2019). 2, 3-Dihydro-3β-methoxy Withaferin-A Lacks Anti-Metastasis Potency: Bioinformatics and Experimental Evidences. Scientific Reports, 9, 17344.

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Western Blot Analysis of Signaling Proteins

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Cell lysates were harvested using RIPA cell lysis buffer. An equal amount (15 ug) of total cellular protein was separated by 12.5% SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore). The membranes were then blocked with 5% non-fat milk for 2 h and incubated overnight with primary antibodies against ERp29 (Abcam), Akt, p-Akt, ERK, p-ERK(Cell Signaling Biotechnology), E-cadherin, vimentin, N-cadherin (Santa Cruz). After incubated with HRP conjugated-secondary antibody for 2 h at room temperature, the proteins were visualized by enhanced chemiluminescence (ECL).

Wu J., Yang Y., Gao S., Jiang H., Wang X.Q., Xiao Y., Chen X.H., Li P, & Xu C.D. (2017). ERp29 inhibits tumorigenicity by suppressing epithelial mesenchymal transition in gastric cancer. Oncotarget, 8(45), 78757-78766.

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