Cells were cultured on glass coverslips with or without poly-L-lysine, then fixed with 4% formaldehyde, permeabilised using 0.15% Triton X-100 in PBS, and blocked with 1% BSA before immunoprobing with anti-vinculin antibodies (catalogue no. 73614, 1:1000, Santa Cruz Biotechnology, 1 h room temperature), and Cy5-secondary anti-rabbit-IgG antibodies (1:1000, AbCam; 1 h room temperature). Samples were counterstained with 20 nM Rhodamine–phalloidin and DAPI, then mounted with Mowiol. Images were acquired using an inverted (Olympus IX71) microscope with 40× NA1.2 oil and 60× NA1.4 oil objectives, DeltaVision software (GE Healthcare) and a Coolsnap HQ2 camera (Photometrics). Maximum projections of deconvolved 0.3-μm z-stacks prepared in Photoshop are presented.
Anti vinculin antibody
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Anti-vinculin antibody is a laboratory reagent used in various research applications. Vinculin is a cytoplasmic protein involved in cell-cell and cell-matrix adhesion. The anti-vinculin antibody can be used to detect and study the localization and expression of vinculin in different cell types and tissues.
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12 protocols using anti vinculin antibody
1
Immunofluorescent Staining of Vinculin
2
Western Blot Analysis of Cellular Proteins
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After treatment and at the indicated time, cells were washed once with PBS and lysed with 6 M urea buffer equipped with protease and phosphatase inhibitors (PMSF, aprotinin, pepstatin, leupeptin, sodium orthovanadate, and sodium pyrophosphate). Protein concentration was measured using Bradford reagent (Sigma-Aldrich, Germany). Equal amounts of protein (50 μg) were loaded into each well of 10% SDS-PAGE for resolving the samples. Subsequently, separated proteins were blotted onto a PVDF membrane (GE Healthcare, Germany). Membranes were then washed with TBS/tween and blocked for 3 h at room temperature with fat-free dried milk before incubation with the indicated primary antibodies at 4 °C for overnight.
Anti-β-actin and anti-vinculin antibodies were purchased from Santa Cruz Biotechnology, whereas anti-mouse and rabbit IgG horseradish peroxidase (HRP)-linked antibodies were purchased from Cell Signaling Technology.
Membranes were developed using Western LightningTM Plus-ECl (Perkin Elmer, Germany) as HRP substrate. Proteins of interest were detected using a Fujifilm LAS-3000 imaging system.
Anti-β-actin and anti-vinculin antibodies were purchased from Santa Cruz Biotechnology, whereas anti-mouse and rabbit IgG horseradish peroxidase (HRP)-linked antibodies were purchased from Cell Signaling Technology.
Membranes were developed using Western LightningTM Plus-ECl (Perkin Elmer, Germany) as HRP substrate. Proteins of interest were detected using a Fujifilm LAS-3000 imaging system.
3
Analysis of Cytoskeletal Regulators
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Anti-phospho-cofilin (Ser-3), anti-cofilin, anti-phospho-MLC (Thr-18/Ser-19), anti-MLC, anti-phopsho-LIMK1 (Thr-508)/LIMK2 (Thr-505), anti-LIMK1, anti-phospho-Src family (Tyr-416), anti-phospho-Src (Tyr-527), anti-Src, anti-phospho-FAK (Tyr-397), anti-FAK, anti-α-actinin, anti-paxillin, anti-talin-1, and anti-tensin-2 antibodies were purchased from Cell Signaling Technology. Anti-PCTK3 and anti-vinculin antibodies were from Santa Cruz Biotechnology. Anti-FLAG (M2) antibody was form Sigma-Aldrich. Anti-Strep antibody was from Qiagen. Anti-Halo antibody was from Promega. Anti-GAPDH antibody was from Wako Pure Chemical Industries. Anti-RhoA and Rac1 antibodies and Alexa-555 conjugated-phalloidin were from Cytoskeleton. Anti-Myc antibody was from Enzo Life Sciences.
4
Western Blot Analysis of Protein Extracts
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Protein was extracted from the tissue samples, and Western blot analysis was performed as described previously [13 (link)]. To confirm the uniformity of protein loading, the blots were incubated with an anti-vinculin antibody (Santa Cruz Biotech, Dallas, TX, USA) or an anti-β-tubulin antibody (Sigma–Aldrich, St. Louis, MO, USA) as a control for total protein extracts. A list of the antibodies used in Western blot analysis is shown in the Supplementary Material (Table S3) .
5
Immunofluorescence Assay for Osteoblast Adhesion
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To investigate the effects of the extraction medium on the cell adhesion and cell morphological features, MC3T3-E1 pre-osteoblasts were fixed with 4% paraformaldehyde for 30 min at 25 °C and then thoroughly washed with PBS (phosphate-buffered saline). Then, the plasma membrane was permeabilized using a solution containing 0.1% Triton X-100 and 2% bovine serum albumin (BSA) to allow the dyes to penetrate the cells and to block the non-specific binding sites. To visualize the actin microfilaments and vinculin, samples were incubated with Alexa Flour-488 phalloidin (Molecular Probes, Eugene, OR, USA) and anti-vinculin antibody (Santa Cruz Biotechnology, Dallas, TX, USA) followed by specific secondary antibody coupled with Alexa Fluor 546 Molecular Probes, Eugene, OR, USA), as previously described [29 (link)]. Finally, a 10 min incubation with 4′,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich, Steinheim, Germany) dye was performed in order to highlight the nuclei.
6
Detecting Cleaved Notch1 and Associated Proteins
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The VAL1744 polyclonal antibody (Cell Signaling Solutions) was used at a dilution of 1:250 to detect cleaved Notch1. The 9E10 monoclonal antibody (Santa Cruz) was used at a concentration of 1:1000 to detect myc-tagged Notch1, JAG1, and Dll4. The β-actin monoclonal antibody (Santa Cruz) was used at a dilution of 1:1000 to detect β-actin as a loading control. The anti-vinculin antibody (Santa Cruz) was used at a dilution of 1:1000
7
Protein Extraction and Quantification from Liver and Adipose Tissue
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Liver samples were homogenized in a lysis buffer using a Potter–Elvehjem homogenizer, and total protein and nuclear extracts were prepared as described previously [7 (link)]. To prepare protein extracts form adipose tissue, the samples were homogenized in a lysis buffer using a TissueLyser LT (Qiagen, Düsseldorf, Germany) at 50 Hz for 10 min, centrifuged at 13,000× g for 15 min at 4 °C, and the supernatant was obtained. Protein content in protein extracts was determined by the Bradford assay [14 (link)]. Western blots were performed using four samples per group, each sample pooled from two animals, as described previously [8 (link)]. To confirm the uniformity of protein loading, blots were incubated with anti-β-actin or anti-β-tubulin antibodies (Sigma-Aldrich, St. Louis, MO, USA), or with an anti-vinculin antibody (Santa Cruz Biotech, Dallas, TX, USA), as a control for total protein extracts, and with an anti-TBP antibody (AbCam, Cambridge, UK) for nuclear protein extracts. A list of antibodies used in Western blot analysis is shown in Table S2 .
8
Protein Extraction and Western Blot Analysis
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Tissue samples and western blot analysis were prepared and performed as described previously.[11 ] Western blots were performed using four samples per group, each sample pooled from two animals. A total of 30 µg of protein extracts (10 µg for PNPLA3 determination) was used. Protein concentrations were determined by the Bradford method.[46 ] To confirm the uniformity of protein loading, blots were incubated with anti‐β‐actin, or anti‐β‐tubulin antibodies (Sigma–Aldrich, St. Louis, MO, USA) or anti‐vinculin antibody (Santa Cruz Biotech, Dallas, TX, USA), as a control for total protein extracts, and with anti‐TBP antibody (AbCam, Cambridge, UK), as a control for nuclear protein extracts. A list of antibodies used in western blot analysis is shown in the Supplementary Material (Table S3 , Supporting Information).
9
Quantitative Western Blot Analysis of Liver Proteins
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Liver samples were homogenized in lysis buffer using a Potter-Elvehjem homogenizer, and total protein and nuclear extracts were prepared as described previously [16 (link)]. Protein content was determined by the Bradford assay [31 (link)]. Western blots were performed using four samples per group, each sample pooled from two animals. A total of 20–30 µg of protein extracts was subjected to SDS-polyacrylamide gel electrophoresis. Proteins were then transferred onto Immobilon polyvinylidene difluoride transfer membranes (Millipore, Billerica, MA, USA) and blocked for 1 h at room temperature with a 5% non-fat milk solution in Tris-buffered saline (TBS) containing 0.1% Tween-20. Membranes were then incubated with specific primary antibodies (see the list of antibodies used in the Supplementary Material, Table S2 ). Detection was performed using the Immobilion Western HRP substrate Peroxide Solution® (Millipore, Billerica, MA, USA). To confirm the uniformity of protein loading, blots were incubated with antivinculin antibody (Santa Cruz Biotech, Dallas, TX, USA) as a control for total protein extracts, and with anti-TBP antibody (AbCam, Cambridge, UK) as a control for nuclear protein extracts.
10
Semi-Quantitative Analysis of VEGFR2 Expression
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The basal expression level of VEGFR2 was determined semi-quantitatively. The HUVECs were harvested to extract total protein for a bicinchoninic acid protein assay (Thermo Fisher Scientific, Inc.) and 30-60 ng protein was used for western blotting. The samples were separated on 8% SDS-PAGE gels and transferred onto a PVDF membrane (Macherey-Nagel) via semi-dry blotting. The membranes were blocked with 5% skim milk (Bio-Rad Laboratories, Inc.) in Tris-buffered saline with 0.1% Tween (TBS-T) or 3% bovine serum albumin (Sigma-Aldrich; Merck KGaA) and stained with either anti-VEGFR2 antibody (cat. no. sc-505, 1:500, Santa Cruz Biotechnology, Inc.) in 2.5% skim milk, or anti-vinculin antibody (cat. no. sc-25336, 1:500, Santa Cruz Biotechnology, Inc.) in TBS-T at 4°C overnight. The secondary antibodies anti-rabbit (cat. no. RPN4301, 1:10,000) and anti-mouse (cat. no. NX931, 1:50,000) coupled to horseradish peroxidase (Amersham; GE Healthcare Life Sciences) were incubated for 2 h at room temperature. The signals were detected with ECL-Prime (GE Healthcare Life Sciences) using ChemiDoc XRS (Bio-Rad Laboratories, Inc.). The expression levels of VEGFR2 were semi-quantitatively analyzed using ImageLab® 5.1 software (Bio-Rad Laboratories, Inc.) with vinculin as a loading control.
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