Diaphragm muscles from at least 5 animals/strain (wild type, mdx, and moAb-Il6r-treated and untreated mdx mice) were homogenized in modified lysis buffer (Tris–HCl, pH 7.5/20 mM, EDTA/2 mM, EGTA/2 mM, sucrose/250 mM, DTT/5 mM, Triton-X/0.1%, PMSF/1 mM, NaF/10 mM, SOV4/0.2 mM, cocktail protease inhibitors/1 × (Sigma) and processed for western blot analysis. Filters were blotted with antibodies against: pStat3 (Tyr705) (cat #9145, Cell Signaling), Stat3 (cat # 9132, Cell Signaling); NFkBp65 (ser536) (cat # 3033, Cell Signaling); and NFkB (cat #4764, Cell Signaling). Signals were captured by ChemiDoc-It® Imaging System (UVP, LLC) and densitometric analysis was performed with VisionWorks®LS Image Acquisition and Analysis Software (UVP, LLC). ELISA assay was performed using either a human (to detect IL6; cat # HS600B) or mouse (to detect Il6, cat # M6000B, and Tnfα, cat # MTA00B) Quantikine® Colorimetric Sandwich ELISAs (R&D Systems), according to manufacturer's protocol. Il2 expression was evaluated on the diaphragm of at least 3 animals/strain and with 2 biological replicates, using a RayBio® Mouse Antibody Array-G series (RayBiotech, Inc.), according to manufacturer's protocol. The intensities of signals were quantified with RayBio® Antibody Array Analysis Tool software.
Visionworks ls image acquisition and analysis software
Manufactured by Analytik Jena
Sourced in United States
VisionWorks LS is an image acquisition and analysis software developed by Analytik Jena. The software is designed to capture, process, and analyze images from various scientific instruments. It provides tools for image optimization, measurement, and data export.
Automatically generated - may contain errors
Lab products found in correlation
Nf kb, by Cell Signaling Technology (2 mentions)
Quantikine colorimetric sandwich elisa, by R&D Systems (2 mentions)
Horseradish peroxidase conjugated secondary antibody, by Santa Cruz Biotechnology (2 mentions)
Liteablot extend, by Euroclone (2 mentions)
Biospectrum imaging system, by Analytik Jena (2 mentions)
Halt protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Polyvinylidene difluoride membrane, by Bio-Rad (2 mentions)
Cocktail protease inhibitors 1x, by Merck Group (2 mentions)
Ne per nuclear and cytoplasmic extraction reagent, by Thermo Fisher Scientific (2 mentions)
Nfkbp65 ser536, by Cell Signaling Technology (1 mentions)
P stat3 tyr705, by Cell Signaling Technology (1 mentions)
Stat3, by Cell Signaling Technology (1 mentions)
Cell lysis buffer, by Cell Signaling Technology (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Merck Group (1 mentions)
Protein assay, by Bio-Rad (1 mentions)
Complete protease inhibitor cocktail, by Roche (1 mentions)
Ecl plus detection system, by Cytiva (1 mentions)
Vinculin, by Merck Group (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Chemiluminescence substrate, by Thermo Fisher Scientific (1 mentions)
15 protocols using visionworks ls image acquisition and analysis software
1
Diaphragm Molecular Profiling in Dystrophy
2
Quantitative Protein Expression Analysis
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After in vitro or in vivo experiments, the cell lysates or tumor tissue homogenates were harvested, and protein concentrations were determined. The expression levels of RAGE, NF-κB/p65, phospho-IκB-α, and MDR1 in the cells and tissues were determined using Western blotting analysis, according to our previous report [22 (link)]. The relative abundances of the signal intensities were quantified using VisionWorks LS Image Acquisition and Analysis Software (version 6.3.3, UVP, Upland, CA, USA).
3
Protein Extraction and Western Blot Analysis
4
Western Blot Analysis of Apoptosis and Autophagy
5
Immunoblotting analysis of cellular proteins
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Whole cell lysates obtained as previously described [13 (link)] and nuclear and cytoplasmic lysates obtained using NE-PER™ Nuclear and Cytoplasmic Extraction Reagents (Thermo Fischer Scientific, Waltham, MA, USA) were immunoblotted as previously described [13 (link)]. Primary antibodies to PARP1 and c-Myc (Cell Signaling Technology, Danvers, MA, USA), XRCC1 and DNA polymerase θ (Abcam, Cambridge, United Kingdom), DNA ligase 3 (BD Biosciences, San Jose, CA, USA), γ-H2AX (S139) (Millipore Sigma, Burlington, MA, USA), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Calbiochem), β-actin (Santa Cruz Biotechnology, Dallas, TX, USA) and vinculin (Sigma-Aldrich) were used. Antibodies to the nuclear protein histone 3 (H3) (Abcam) and the cytoplasmic protein growth factor receptor-bound protein 2 (GRB2) (BD Biosciences) were used as controls in analyses of nuclear and whole cell protein levels. Densitometry was performed with VisionWorks LS Image Acquisition and Analysis Software (UVP, Upland, CA, USA). Protein levels were normalized to β-actin. Triplicate experiments were performed.
6
Protein Expression Analysis in Ocular Cells
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Mouse eyecups or ARPE-19 Cells were lysed in RIPA buffer with protease inhibitor cocktail, PMSF and sodium orthovanadate (Santa Cruz Biotechnology) and sonicated by ultrasound (on ice). Protein concentration was quantified using BCA kit (Pierce Biotechnology, Inc., Rockford, IL, United States). Twenty-five micrograms of protein were resolved by SDS-PAGE gel and electro-transferred to nitrocellular membranes. After blocking with 5% non-fat milk, membranes were blotted overnight at 4°C with following primary antibodies: anti-Nrf2 (1:1000; Santa Cruz Biotechnology), anti-XBP1 (1:500; Santa Cruz Biotechnology), anti-Catalase (1:2000; Sigma-Aldrich), anti-SOD1 (1:2000; Abcam), anti-SOD2 (1:1000; Assay Designs, MI, United States) and anti-β-actin (1:5,000; Abcam). After incubation with HRP-conjugated secondary antibodies, membranes were developed with chemiluminescence substrate (Thermo Fisher Scientific, Rockford, IL, United States: #34076) using Vision Works LS image acquisition and analysis software (UVP, Upland, CA, United States), and bands were semi-quantified by densitometry.
7
Saquinavir Induces Protein Ubiquitination in HeLa Cells
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After 2 h of treatment with 40, 60 and 80 µM saquinavir, whole HeLa cell protein extracts were prepared in 150 mM NaCl, 1% Nonidet-40, 50 mM Tris-HCl (pH 7.5) and Halt Protease Inhibitor Cocktail (Thermo Fisher Scientific, Inc.). Cell extracts (20 µg) were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (NuPAGE® Novex® 4–12% Bis-Tris gels; Thermo Fisher Scientific, Inc.) and blotted onto polyvinylidene difluoride membranes (Bio-Rad Laboratories, Inc., Hercules, CA, USA). α-Tubulin was used as a protein loading control. Following blocking in Tris-buffered saline containing 5% non-fat milk, the blots were incubated with primary antibodies against α-tubulin (dilution, 1:20,000; T5168; Sigma-Aldrich; Merck Millipore) or ubiquitin (dilution, 1:200; P4D1; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) at 4°C for 12 hours, followed by incubation with horseradish peroxidase-conjugated secondary rabbit anti-mouse IgG (dilution, 1:10,000; catalog no., A9044; Sigma-Aldrich; Merck Millipore) at room temperature for 1 h. Signals were detected on a BioSpectrum Imaging System (UVP, Inc., Upland, CA, USA) with the LiteAblot® EXTEND (Euroclone SpA). Images were processed with VisionWorks® LS Image Acquisition and Analysis software version 7.0.1 (UVP, Inc.).
8
Western Blot Analysis of CREB Activation
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The cells were incubated for 12 h in the presence or absence of 2.5 μg/ml of DMGF. Then, the cells were collected and lysed in ice-cold RIPA lysis buffer. The protein concentration of the cell lysate was estimated with the Bradford protein assay using BSA as the standard. Total proteins (50−60 μg) were separated by SDS-PAGE using a 10% polyacrylamide gel and then transferred onto a nitrocellulose membrane. The membrane was blocked with 5% skim milk in phosphate buffered saline with Tween 20 (0.05% v/v Tween-20 in PBS, pH 7.2) for 1 h. The membranes were then incubated with anti-CREB antibody (1:1,000; GeneTex, Irvine, CA, USA) and anti-pCREB antibody (1:1000; Abcam, Cambridge, CA, USA) at 4°C overnight, followed by incubation with a horseradish peroxide-linked secondary antibody (1:10000; GeneTex, Irvine, CA, USA). The protein bands were visualized using the ChemiLucent ECL Detection System (Millipore, Billerica, MA, USA) and the Biospectrum AC Imaging System (UVP, Upland, CA, USA). The intensities of the chemiluminescence signal were quantified using the UVP VisionWorks LS Image Acquisition and Analysis Software (UVP, Upland, CA, USA).
9
Western Blot Analysis of FOXM1 Protein
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Whole protein extracts from cells at 24 h following siRNA transfection or untransfection were lysed in NaCl, 1% Nonidet‑40, 50 mM Tris‑HCl (pH 7.5) and Halt Protease Inhibitor Cocktail (Thermo Fisher Scientific), and protein concentrations were determined using a Bio-Rad protein assay system (Bio-Rad). Equivalent amounts of proteins were separated by SDS-PAGE, and then transferred to polyvinylidene difluoride membranes (Bio-Rad). After being blocked in Tris-buffered saline (TBS) containing 5% non-fat milk, the blots were incubated with the following primary antibodies: anti-FOXM1 (clone sc-502, 1:200 dilution, Santa Cruz Biotechnology, Santa Cruz, CA), and anti-β-actin ((20–33), 1:200 dilution) (Sigma-Aldrich, Saint Louis, Missouri, USA), at 4 °C for 12 h, followed by incubation with horseradish peroxidase-conjugated secondary (anti-mouse or anti-rabbit) IgGs at room temperature for 1 h. Signals were detected on BioSpectrum® Imaging System (UVP, LLC, Upland, CA, USA) with the LiteAblot® EXTEND (EuroClone). Images were processed with VisionWorks® LS Image Acquisition and Analysis software (version 7.0.1, UVP, LLC).
10
Analyzing Dystrophic Diaphragm Muscle Signaling
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Diaphragm muscles from at least five animals/strain (wild-type, mdx and mdx/IL6 mice) were homogenized in modified lysis buffer [Tris–HCl, ph 7.5/20 mm , EDTA/2 mm , EGTA/2 mm , sucrose/250 mm , DTT/5 mm , Triton-X/0.1%, PMSF/1 mm , NaF/10 mm , SOV4/0.2 mm , cocktail protease inhibitors/1X (Sigma)]. Filters were blotted with antibodies against: Pax-7, Desmin, NFkBp65 (ser536), NFkB (Cell Signaling); GAPDH (Santa Cruz). Signals were captured by ChemiDoc-It® Imaging System (UVP, LLC) and densitometric analysis was performed with VisionWorks®LS Image Acquisition and Analysis Software. Elisa assay was performed to detect murine and transgenic IL-6 using Quantikine® Colorimetric Sandwich ELISAs (R&D Systems), according to manufacturer's protocol.
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