HeLa cells were transiently co-transfected with LOVTRAP plasmids for 20 hours before lysis in RIPA buffer. The cell extracts were adjusted to the same amount of total cellular protein (50 μg), and electrophoresed in a 4% to 15% gradient polyacrylamide gel. After electrophoretic transfer to a PVDF membrane at 1.0A for 30 min, the membranes were blocked with TBST (10mM Tris-HCl, pH7.5, 150mM NaCl, 0.05%Tween-20) containing 5% BSA for 1 hour at room temperature. The primary antibodies (mouse monoclonal anti-Rac1 antibody from Cytoskeleton, Inc., Cat. # ARC03, 1:500 dilution28 ; mouse monoclonal anti-GFP Antibody from Clontech, Cat# 632381, 1:1000 dilution29 ; and mouse monoclone anti-human vinculin antibody Clone h-VIN1 from Sigma, Cat# V9131, 1:200 dilution30 ) in TBST were placed on the membrane and incubated at 4°C overnight. After three washings with TBST over 5 min, the second antibody (Anti-mouse IGG, conjugated with DyLight680, from Cell Signaling, Cat#5470P, 1:10,000 dilution31 (link)) was applied. After three washings with TBST over 5 min, the membrane was scaned by the Odyssey® Infrared Imaging System (LI-COR Biosciences, Lincoln, NE).
Clone hvin 1
Manufactured by Merck Group
Sourced in United States
The Clone hVIN-1 is a laboratory equipment product designed for specific research applications. It is a tool used in various scientific and medical research fields. The core function of this product is to facilitate the cloning and analysis of the human VIN-1 gene. No further details or interpretations about its intended use are provided.
Automatically generated - may contain errors
Lab products found in correlation
Mouse monoclonal anti rac1 antibody, by Cytoskeleton (2 mentions)
Dylight680, by Cell Signaling Technology (2 mentions)
Mouse monoclonal anti gfp antibody, by Takara Bio (2 mentions)
Odyssey infrared imaging system, by LI COR (2 mentions)
Bca assay, by Thermo Fisher Scientific (2 mentions)
Clone d9e, by Cell Signaling Technology (1 mentions)
Clone c67e7, by Cell Signaling Technology (1 mentions)
Goat anti mouse igg dylight 800, by Thermo Fisher Scientific (1 mentions)
Goat anti rabbit igg dylight 800, by Thermo Fisher Scientific (1 mentions)
Odyssey fluorescence imager, by LI COR (1 mentions)
Supersignal west femto maximum sensitivity substrate, by Thermo Fisher Scientific (1 mentions)
Dl dithiothreitol, by Merck Group (1 mentions)
Anti α tubulin antibody, by Abcam (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Dojindo Laboratories (1 mentions)
Rhodamine conjugated phalloidin, by Thermo Fisher Scientific (1 mentions)
710 elyra ps 1, by Zeiss (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Cell lysis buffer, by Cell Signaling Technology (1 mentions)
Sc 46, by Santa Cruz Biotechnology (1 mentions)
Complete protease inhibitor cocktail, by Roche (1 mentions)
10 protocols using clone hvin 1
1
Rac1 and Vinculin Detection in HeLa Cells
2
Phospho-Proteomics of Activated T-cells
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T-cells from co-culture experiments (1 × 105 T-cells seeded) were harvested with one part of 1× PBS and one part of Novex® buffer (Thermo Fisher Scientific) containing 3% dl -dithiothreitol (Sigma-Aldrich). Samples were run on 10% SDS PAGE gels and then blotted on an Amersham protan nitrocellulose transfer membrane (Sigma-Aldrich). Phospho-protein phosphatase 2A (pPP2A) (1:7,000, clone E155; Abcam, Cambridge, UK), protein phosphatase 2A (PP2A) (1:1,000), pAkt (Ser 473, 1:2,000, clone D9E), pAkt (Thr 308, 1:1,000, clone C31E5E), Akt (1:1,000, clone C67E7, all Cell signaling technology, Danvers, MA, USA), and vinculin (clone hVIN-1, 1:40,000 Sigma-Aldrich) protein expressions were determined by immunoblotting of CD3+ T-cells at intervals up to 96 h after allogeneic stimulation with DCs. Western blots for pPP2A and pAKt (Ser473 and Thr308) were developed by chemiluminescence imaging using the Super signal west femto maximum sensitivity substrate (Thermo Fisher Scientific) and chemiluminescent detection films (Sigma-Aldrich). Western blots for PP2A, Akt, and vinculin were developed by fluorescence imaging using Goat-anti-mouse IgG Dylight 800 and Goat-anti-rabbit IgG Dylight 800 (both Thermo Scientific) on an Odyssey Fluorescence imager (Licor, Licoln, NE, USA). Western blots were analyzed by densitometry using the ImageJ software.
3
Visualization of Cytoskeletal Proteins in Macrophages
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Treated cells (U937 macrophages or BMDMs) were fixed with 4% paraformaldehyde (Sigma, USA) and washed and permeabilized with 0.5% Triton X-100 and rinsed 3 times with PBS. Nonspecific binding sites were blocked by adding PBS with 1% BSA. The adhesion protein, vinculin, was labelled by incubation at 4 °C, overnight with mouse monoclonal anti-vinculin primary antibody, clone hVIN-1 (1:400, Sigma, USA). Cells were then washed and incubated for 1 h at room temperature in Alexa 488 conjugated anti-Mouse IgG as Secondary Antibody (1:1000, Invitrogen, USA). In separate samples, cellular α-tubulin and acetylated α-tubulin were labelled using rabbit polyclonal anti-α tubulin antibody (1:200, Abcam, UK) and mouse anti-acetylated tubulin, clone 611B-1 (1:2000; Sigma-Aldrich, USA) respectively with Alexa 488 secondary antibodies as above.
To simultaneously label the F-actin, the samples were incubated for 30 mins with 200 μl of rhodamine-conjugated phalloidin (Molecular Probes, USA) in a humidified chamber at room temperature in the dark. Cell nuclei were stained by incubation with 5 μg/ml DAPI (Dojindo, USA) followed by two PBS rinses. Slides were then visualized with a fluorescence microscope (SP2, Leica, Germany) with ×63 objective. Slides were also imaged on a microscope (710 ELYRA PS.1, Zeiss, Germany) with a ×63/1.4NA objective for structural illumination microscopy.
To simultaneously label the F-actin, the samples were incubated for 30 mins with 200 μl of rhodamine-conjugated phalloidin (Molecular Probes, USA) in a humidified chamber at room temperature in the dark. Cell nuclei were stained by incubation with 5 μg/ml DAPI (Dojindo, USA) followed by two PBS rinses. Slides were then visualized with a fluorescence microscope (SP2, Leica, Germany) with ×63 objective. Slides were also imaged on a microscope (710 ELYRA PS.1, Zeiss, Germany) with a ×63/1.4NA objective for structural illumination microscopy.
4
Western Blot Analysis of Neuronal Proteins
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CNS tissues were lysed in cell lysis buffer (Cell Signaling), supplemented with cOmplete Protease Inhibitor Cocktail and PhosSTOP Phospatase inhibitors (both Roche), for 30 min, sonicated and centrifuged at 14`000 x g at 4°C for 30 min. After BCA assay (Thermo Scientific), proteins were blotted and detected with the following antibodies: mouse anti-Vinculin (loading control, 1: 20`000, clone hVIN-1, Sigma), rabbit polyclonal anti-junB (1:250, Santa Cruz, sc-46) and mouse anti-c-jun (1:1`000, clone 3/Jun, BD Transduction Laboratories).
5
Western Blot Analysis of EBNA1 in Transfected HEK293T Cells
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Transfected HEK293T cells were detached, centrifuged, and washed in ice-cold DPBS. Cell pellets were lysed in RIPA buffer (Sigma) containing 1% v/v protease inhibitor cocktail (Sigma). The protein concentration of clarified lysates was determined using a BCA assay (Thermo Fisher). Lysates were denatured, and 20 μg were loaded into SDS PAGE gels. Proteins were transferred onto nitrocellulose membranes (Thermo Fisher) and probed for EBNA1 (Merck, clone 1H4, monoclonal, cat MABF2800, dilution 1:1000) and a loading control, Vinculin (Sigma, clone hVIN-1, monoclonal, cat V9264, dilution 1:5000). Secondary antibodies (Life Technologies, Goat anti-Rat IgG A18868, polyclonal, and Abcam Rabbit Anti-Mouse IgG H&L (Alkaline Phosphatase) ab6729, dilutions 1:10000) were detected using the Novex™ AP Chromogenic Substrate (Invitrogen). Image J48 was used for densitometry analysis. To obtain a normalisation factor specific to each lane, the Vinculin signal for each lane was normalised against the lane with the strongest signal. This was then multiplied by the EBNA1 signal of the same lane to get a normalised EBNA1 experimental signal. Finally, to control for inter-blot variability, normalised EBNA1 values were divided once more against the average normalised signal of each lane present within the blot (except the negative control).
6
Rac1 and Vinculin Detection in HeLa Cells
7
Cytoplasmic and Nuclear Fractionation Protocol
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Cytoplasmic and nuclear fractions were prepared as previously described in detail (Suzuki et al., 2010 (link)). In brief, cells were washed with PBS (37°C) and lysed for 10 min on ice with cytoskeleton lysis buffer (CLB; 0.5% Triton X-100, 10 mM 1,4-piperazinediethanesulfonic acid [pH 7.4], 150 mM NaCl, 5 mM EGTA, 5 mM glucose, 5 mM magnesium chloride, supplemented with 0.5 mM sodium fluoride, 1 mM sodium orthovanadate, and protease inhibitors cocktail). Afterward the Triton soluble fraction, corresponding to the cytosolic fraction, was collected and clarified at 10,000 × g for 10 min at 4°C. The cell monolayer was then washed twice with ice-cold cell lysis buffer and incubated with RIPA buffer for 15 min on ice. After the cells were scraped off, the RIPA buffer (Triton-insoluble or cytoskeletal fraction) was clarified at 12,000 × g for 10 min at 4°C. Both fractions were resolved by SDS-PAGE and probed by western blotting with antibodies against TAZ (1:1,000; V386, Cell Signaling Technology), actin (1:10,000; clone AC-74, Sigma-Aldrich), tubulin (clone YL1/2 [Gamper et al., 2016 (link)]), and vinculin (1:1,000; clone hVin-1, Sigma-Aldrich).
8
Quantitative Fibrocystin Western Blot Analysis
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MDCKII cells were lysed directly in 65 °C prewarmed 2x Laemmli-Buffer (150 µL/75 cm²) and analyzed using 4–15% gradient gels (4–15% Mini-PROTEAN TGX Precast Protein Gels, Biorad, Hercules, CA, USA) and pre-stained molecular weight markers (PageRuler Prestained Protein Ladder (#26616), 10 to 180 kDa, and HiMark Pre-stained Protein Standard (LC5699), 30 to 460 kDa; Thermo Fisher Scientific). Transfer was performed in Towbin Buffer (0.0375% SDS, without methanol). For immunodetection, rabbit anti-fibrocystin (1:200, C-20, sc-49671; Santa Cruz Biotechnology), mouse anti-vinculin (1:1000, clone hVIN-1; Sigma-Aldrich), and rabbit anti-myosin IIa (1:500, M8064, Sigma-Aldrich) were used. Secondary antibodies, IRDye 800CW goat anti-rabbit IgG (H+L) (1:5000 or 1:10,000) and IRDye 680RD goat anti-mouse IgG (H+L) (1:5000) allowed quantitative fluorescence detection via the Odyssey Fc Imaging System (antisera and detection system, LI-COR Biotechnology, Bad Homburg, Germany). Signal intensities were determined by using Image Studio Software (5.2) and FPC protein normalized using geometric means of vinculin and myosin II signals to adjust protein load.
9
Protein Extraction and Western Blot Analysis
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Cells were harvested by centrifugation at 11,000 rcf in 4 °C for 10 s, quick-frozen in liquid nitrogen, thawed on ice, incubated in lysis buffer (20 mM Tris-HCl, 150 mM NaCl, 1% v/v Triton X-100, 0.5 mM EDTA, 1 mM Na3VO4, 10 mM NaF, 2.5 mM Na4P2O7, 0.5% sodium deoxycholate, protease inhibitor (Sigma, #P2714)) for 30 min and vortexed repeatedly. The cell lysates were then cleared from cell debris by centrifugation at 20,000 rcf for 15 min. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blot were performed according to standard protocol. The antibodies used for protein detection were mouse anti-phospho-tyrosine (Merck-Millipore, Darmstadt, Germany; clone 4G10, #05-1050); rabbit anti-OPA1 (described previously37 (link)); mouse anti-ACTB (Sigma; clone AC-74, #A5316); and mouse anti-VCL (Sigma; clone hVIN-1, #V9131).
10
Western Blot Analysis of Cellular Proteins
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Cells were pelleted and lysed using ice-cold RIPA buffer supplemented with protease and phosphatase inhibitor cocktail (Thermo Fisher). Protein concentration was determined using DC protein assay (Bio-Rad). Equal amounts of protein extracts (40–60 μg) were loaded and separated by SDS–PAGE, and then transferred to polyvinylidene fluoride (PVDF) membranes. Membranes were blocked for 45 min at room temperature with 5% non-fat milk (Bio-Rad) in TBS plus 0.05% Tween 20, followed by overnight incubation at 4 °C with primary antibodies against BRCA1 (1:500, Abcam, # ab238983), STING (1:1000, clone D2P2F, Cell Signaling Technology, # 13647S), or VINCULIN (1:2000, clone hVIN-1, Sigma, # V9131). Blots were then incubated with fluorescently labeled anti-mouse IgG (1:2000, Rockland Immunochemicals, # RL610-145-002) or anti-rabbit IgG (1:2000, Molecular Probes, # A-21109) at room temperature for 1 h. Western blots were visualized on an Odyssey scanner (LI-COR). Uncropped blots are provided in the Source Data file.
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