Anti villin

Manufactured by Santa Cruz Biotechnology

Sourced in United States

Anti-villin is a laboratory reagent used in scientific research. It is a specific antibody that recognizes and binds to the villin protein, which is a key component of the cytoskeleton in certain cell types. Anti-villin can be used in various experimental techniques, such as Western blotting, immunohistochemistry, and immunocytochemistry, to detect and analyze the presence and distribution of villin in biological samples.

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20 protocols using anti villin

Isolation and Analysis of Mouse Colonic Epithelium

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Mouse colonic mucosa was collected by scraping proximal and distal regions as previously described.^{20 (link)} Tumors were collected separately. Mouse epithelial cells were lysed in lysis buffer (1% Triton X-100, 150 mM NaCl, 10 mM Tris pH 7.4, 1 mM EDTA, 1 mM EGTA pH 8.0, 0. 2 mM sodium orthovanadate, protease inhibitor cocktail). Immunoblotting was performed with primary antibodies: anti-AvrA (custom-made in Sub lab^{13 (link)}), anti-β-catenin (BD, Transduction Laboratories), anti-Bmi 1 (Abcam), and anti-villin (Santa Cruz Biotechnology Inc.), anti-GSK-3β (BD), anti-phospho-GSK-3β (Ser9), anti-phospho-β-catenin (Ser552), anti-phospho-β-catenin (Ser33, Ser37, Thr41) and anti-phospho-Akt (T308) (Cell Signaling, Beverly, MA, USA), or anti-β-actin (Sigma-Aldrich) antibodies, and visualized by enhanced chemiluminescence.^{43 (link), 44 (link)}

Lu R., Wu S., Zhang Y.G., Xia Y., Liu X., Zheng Y., Chen H., Schaefer K.L., Zhou Z., Bissonnette M., Li L, & Sun J. (2014). Enteric bacterial protein AvrA promotes colonic tumorigenesis and activates colonic beta-catenin signaling pathway. Oncogenesis, 3(6), e105-.

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Immunoblotting of Protein Complexes

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Immunoprecipitated protein was lysed in RIPA buffer (Beyotime, P0013B) and loaded into 12% SDS-PAGE gels to be transferred onto a PVDF membrane (Millipore, IPVH00010). The primary antibody at dilutions recommended by the suppliers was anti-ASGRP1 (Santa Cruz Biotechnology, sc-52623), anti-CD63 (Santa Cruz Biotechnology, sc-5275), anti-CD9 (Santa Cruz Biotechnology, sc-13118), anti-villin (Santa Cruz Biotechnology, sc-58897), anti-GFP (Cell Signaling Technology, 2956S), anti-mCherry (Abcam, ab167453), anti-β-actin (Cell Signaling Technology, 4,970).

Li W., Wang J., Yin X., Shi H., Sun B., Ji M., Song H., Liu J., Dou Y., Xu C., Jiang X., Li J., Li L., Zhang C.Y, & Zhang Y. (2022). Construction of a mouse model that can be used for tissue-specific EV screening and tracing in vivo. Frontiers in Cell and Developmental Biology, 10, 1015841.

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Western Blot Analysis of Protein Expression

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Cell lysates were prepared using RIPA buffer (Sigma-Aldrich; R0278) supplemented with protease inhibitors (Roche; 04693124001). Samples were centrifuged at 20,000 × g for 15 min to remove cell debris. The resulting supernatant was boiled with Laemmli sample buffer for 5 min. Samples were then loaded on a 4%–12% NuPAGE gradient gel (Invitrogen; NP0322BOX). Gels were transferred onto a nitrocellulose membrane at 30V for 18 h. Membranes were blocked with 5% dry milk diluted in 1X PBS containing 0.1% Tween 20 (PBS-T) for 2 h at room temperature. The membranes were incubated with primary antibody diluted in 1X PBS-T containing 1% BSA overnight at 4C. Primary antibodies used were anti-MISP (Thermo Scientific; PA5–61995), anti-villin (Santa Cruz; sc-66022), anti-GAPDH (Cell Signaling; 2118), anti-β-actin (Sigma-Aldrich; A5316). Membranes were then washed with 1X PBS-T and incubated with secondary antibodies for 1 h at room temperature. Secondary antibodies used were IRdye 800 donkey anti-rabbit (LI-COR; 926–32213) or donkey anti-mouse (LI-COR; 926–32212). Membranes were washed with 1X PBS-T and imaged using the Odyssey CLx infrared scanner (LI-COR). Images were processed using the FIJI software (NIH). Protein expression levels were normalized to GAPDH.

Morales E.A., Arnaiz C., Krystofiak E.S., Zanic M, & Tyska M.J. (2022). Mitotic Spindle Positioning (MISP) is an actin bundler that selectively stabilizes the rootlets of epithelial microvilli. Cell reports, 39(3), 110692.

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Comprehensive Histopathological and Immunofluorescence Analysis

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Histopathological examinations were performed after Mayer’s H&E staining. Immunofluorescence staining was performed on formalin-fixed paraffin-embedded tissue by using the Tyramide Signal Amplification (TSA) Cy3 system (Perkin&Elmer) according to the manufacturer’s protocol. The following primary antibodies were used: Anti-Villin (Santa Cruz), anti-β-Catenin (Cell Signaling), anti-Caspase-8 (Cell Signaling) and anti-Cleaved Caspase-8 (Cell Signaling). A biotinylated anti-rabbit secondary antibody from Dianova was used. Nuclei were counterstained with Hoechst 33342 (Invitrogen). For cell death analysis the In Situ Cell Death Detection Kit for TUNEL (TdT-mediated dUTP nick end labelling) from Roche was used. Bright-field and fluorescence pictures were taken by using the DMI4000 B microscope (Leica) in combination with a LEICA DFC360 FX or LEICA DFC420C camera.

Ruder B., Günther C., Stürzl M., Neurath M.F., Cesarman E., Ballon G, & Becker C. (2020). Viral FLIP blocks Caspase-8 driven apoptosis in the gut in vivo. PLoS ONE, 15(1), e0228441.

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Immunoblotting Analysis of Epithelial Tight Junction Proteins

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The organoid cells were rinsed three times in ice‐cold HBSS and then suspended in ice‐cold HBSS. The organoid cells were then spun down at 900 rpm for 10 min at 4°C. Next, using a pipette to aspirate the PBS at the top, the organoid cells were lysed in lysis buffer (1% Triton X‐100, 150 mmol/L NaCl, 10 mmol/L Tris pH 7.4, 1 mmol/L EDTA, 1 mmol/L EGTA pH 8.0, 0.2 mmol/L sodium orthovanadate, protease inhibitor cocktail) and then sonicated. The protein concentration was then measured. Next, equal amounts of protein (20 μg/well) were separated by SDS‐polyacrylamide gel electrophoresis, transferred to nitrocellulose, and immunoblotted with primary antibodies. The following antibodies were used: anti‐ZO1, anti‐occludin, anti‐Claudin‐2, anti‐Claudin‐7 (Invitrogen, Carlsbad, CA), anti‐Villin, anti‐p‐P65, anti‐P65, anti‐Iκβα (Santa Cruz, Dallas, TX), anti‐β‐actin (Sigma‐Aldrich, St. Louis, MO), and anti‐p‐Iκβα (Cell Signal, Beverly, MA). Following the primary antibody step, the nitrocellulose membranes were incubated with secondary antibodies and visualized by ECL.

Zhang Y., Wu S., Xia Y, & Sun J. (2014). Salmonella‐infected crypt‐derived intestinal organoid culture system for host–bacterial interactions. Physiological Reports, 2(9), e12147.

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Colonic Epithelial Cell Protein Analysis

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Mouse colonic epithelial cells were collected by scraping the tissue from the colon of the mouse, including the proximal and distal regions. The cells were sonicated in lysis buffer (10 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, pH 8.0, 1% Triton X-100) with 0.2 mM sodium ortho-vanadate, and protease inhibitor cocktail. The protein concentration was measured using the BioRad Reagent (BioRad, Hercules, CA, USA). Cultured cells were rinsed twice with ice-cold HBSS, lysed in protein loading buffer (50 mM Tris, pH 6.8, 100 mM dithiothreitol, 2% SDS, 0.1% bromophenol blue, 10% glycerol), and then sonicated. Equal amounts of protein were separated by SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and immunoblotted with primary antibodies. The following antibodies were used: anti-Claudin-5 (Invitrogen, 35-2500, Carlsbad, CA, USA), anti-Claudin-7 (Invitrogen, 34-9100, Carlsbad, CA, USA), anti-VDR (Santa Cruz Biotechnology, SC-13133, Dallas, TX, USA), anti-Villin (Santa Cruz Biotechnology, SC-7672 Dallas, TX, USA), or anti-β-actin (Sigma-Aldrich, A5316, St. Louis, MO, USA) antibodies and were visualized by ECL (Thermo Fisher Scientific, 32106, Waltham, MA, USA). Membranes that were probed with more than one antibody were stripped before re-probing.

Zhang Y., Garrett S., Carroll R.E., Xia Y, & Sun J. (2022). Vitamin D Receptor Upregulates Tight Junction Protein Claudin-5 against Colitis-Associated Tumorigenesis. Mucosal immunology, 15(4), 683-697.

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Tissue Cell Type and Organelle Immunostaining

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Tissue sections were blocked with 5% BSA with 0.5%Triton X-100 for 45 min, and then incubated with primary antibodies overnight. For tissues cell types analysis, anti-Villin (Santa Cruz Biotechnology, sc-58897), anti-Alblumin (abcam, ab207327), anti-SP-C (Santa Cruz Biotechnology, sc-518029), anti-nephrin (Santa Cruz Biotechnology, sc-377246) were used, and for sEV related organelles, anti-lamp2 (Santa Cruz Biotechnology, sc-18822), anti-Rab7 (Santa Cruz Biotechnology, sc-376362), anti-EEA1 (Santa Cruz Biotechnology, sc-137130) were used. After incubation with primary antibody, samples were incubated with appropriate secondary antibody (Invitrogen, Goat anti-Rabbit IgG (H + L)-Alexa Fluor Plus647, A32733 and Goat anti-Rabbit IgG (H + L)-Alexa Fluor Plus647, A32728) for 1 h and then Hoechst (Invitrogen, H1399) was used for nuclei staining.

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Immunofluorescence Staining of Cytoskeletal Proteins

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Cells grown on a glass coverlips were fixed with 4% paraformaldehyde (EMS; 15710) in 1X PBS for 15 min at 37°C. Fixed cells were washed with 1X PBS, and permeabilized with 0.1% Triton X-100 in 1X PBS for 15 min at room temperature. Cells were washed with 1X PBS and blocked with 5% Bovine Serum Albumin (BSA) in 1X PBS for 2 h at room temperature. Cells were washed and incubated with primary antibodies overnight at 4°C. Primary antibodies used were anti-MISP (Thermo Scientific; PA5–61995), anti-villin (Santa Cruz; sc-66022), anti-ezrin (CST; 3145). Cells were washed with 1X PBS four times for 5 min and incubate with secondary antibodies. Goat anti-rabbit Alexa Fluor 488 F(ab’)2 Fragment (Molecular Probes; A11070), goat anti-mouse Alexa Fluor 568 F(ab’)2 Fragment (Molecular Probes; A11019), Alexa Fluor 568-phalloidin (Invitrogen; A12380), Wheat Germ Agglutinin 405M (WGA) (Biotium; 29028–1), DRAQ5 (Thermo Scientific; 62251). Cells were washed again with 1X PBS and mounted on glass slides using ProLong Gold (Invitrogen; P36930).

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Immunofluorescence Staining of Epithelial Markers

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Immunofluorescence analysis was performed as described previously (Chen et al., 2020b (link)). Fluorescence images were obtained with a microscope (NIS-Elements, Nikon, Tokyo, Japan). The following antibodies and reagents were used: anti-ZO-1, anti-claudin-1, and anti-Ki67 (#NB500-170, Novus Biologicals, Littleton, CO, USA), anti-Villin (#SC-58897, Santa Cruz Biotechnology); anti-p-JAK2, anti-p-STAT3, and FITC-conjugated secondary antibody (#115-545-003, Jackson Laboratory, Jackson, MS, USA); Cy3-conjugated secondary antibody (#111-165-045, Jackson Laboratory); and 4′,6-diamidino-2-phenylindole (DAPI, Sigma–Aldrich).

Chen M.J., Zhou J.Y., Chen Y.J., Wang X.Q., Yan H.C, & Gao C.Q. (2021). The in ovo injection of methionine improves intestinal cell proliferation and differentiation in chick embryos by activating the JAK2/STAT3 signaling pathway. Animal Nutrition, 7(4), 1031-1038.

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Western Blot Analysis of Intestinal Epithelial Cells

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Mouse intestinal epithelial cells were lysed in lysis buffer (1% Triton X-100, 150 mM NaCl, 10 mM Tris, pH 7.4, 1 mM EDTA, 1 mM EGTA, pH 8.0, 0.2 mM sodium orthovanadate, and protease inhibitor cocktail), and the protein concentration was measured. The cells were rinsed two times in ice-cold HBSS, lysed in protein loading buffer (50 mM Tris, pH 6.8, 100 mM dithiothreitol, 2% SDS, 0.1% bromphenol blue, and 10% glycerol), and sonicated. Equal amounts of protein were separated by SDS-PAGE, transferred to nitrocellulose membranes, and subjected to immunoblotting with primary antibodies. The following antibodies were used: anti-Wnt1 (Cell Signaling), anti-villin (Santa Cruz Biotechnology, Santa Cruz, CA), and anti–β-actin (Sigma-Aldrich, Milwaukee, WI). Bands were quantified using Kodak MI software (version 4.0.3).

Wang J., Lu R., Fu X., Dan Z., Zhang Y.G., Chang X., Liu Q., Xia Y., Liu X, & Sun J. (2018). Novel Regulatory Roles of Wnt1 in Infection-Associated Colorectal Cancer. Neoplasia (New York, N.Y.), 20(5), 499-509.

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