Cell cultures were rinsed with PBS, pH=7.4, and lysed in RIPA buffer as previously described [15 (link)]. After protein concentration quantification with a Micro BCA protein assay kit (Thermo Scientific), an equal quantity of protein was separated by SDS-PAGE and transferred to PVDF-membrane (Immobilon Millipore). After 2 hours blocking with 5% milk and 1% Tween in PBS, the membrane was exposed to primary antibodies (in blocking solution) detecting Cx40 (Cx40-A; Alpha-Diagnostics lot #175455A8.6; 1/500), NFκB (NFκB p65 (A) sc-109; Santa Cruz; 1/1000), P-NFκB (Phospho-NFκB (S536)(93H1); Cell Signaling; 1/1000), IκBα (IκBα L35A5; Cell Signaling; 1/1000), P-IκBα (Phospho-IκB-α (Ser32)(14D4); Cell Signaling; 1/1000) and GAPDH (Millipore MAB374; lot #2388833; 1/30000) as loading control. Revelation was performed by incubating the membrane for 1 hour at RT with secondary horseradish peroxidase-conjugated antibodies (Jackson Immunoresearch; 1/5000) and followed by ECL detection (Millipore) using the Fuji LAS3000 (Fujifilm) and ImageQuant LAS 4000 software. Band intensities were thereafter quantified using ImageJ software.
Fuji las3000
Manufactured by Fujifilm
Sourced in Japan, United States
The Fuji LAS3000 is a lab equipment product designed for advanced image analysis and documentation. It features a high-resolution imaging system and versatile software capabilities for a range of applications.
Automatically generated - may contain errors
Lab products found in correlation
Micro bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Mab374, by Merck Group (1 mentions)
Phospho iκbα ser32 14d4, by Cell Signaling Technology (1 mentions)
Cx40 a, by Alpha Diagnostic (1 mentions)
Nitrocellulose membrane, by GE Healthcare (1 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Lumi light substrate, by Roche (1 mentions)
Hrp conjugated secondary antibody, by Agilent Technologies (1 mentions)
Nitrocellulose membrane, by Cytiva (1 mentions)
Ab13518, by Abcam (1 mentions)
Anti gapdh, by Cell Signaling Technology (1 mentions)
Cellytic mt mammalian tissue lysis extraction reagent, by Merck Group (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Multi gauge software, by Fujifilm (1 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Image reader las 3000, by Fujifilm (1 mentions)
Human angiogenesis array kit, by R&D Systems (1 mentions)
Rat cytokine array kit, by R&D Systems (1 mentions)
Panel a, by R&D Systems (1 mentions)
Cl xposure film, by Thermo Fisher Scientific (1 mentions)
8 protocols using fuji las3000
1
Western Blot Analysis of Signaling Proteins
2
Quantifying PD-L1 Expression in Transfected Cells
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For the quantification of CD274 (PD‐L1) protein expression, total protein from transfected and control cells was extracted and quantified using the Pierce BCA protein assay kit (Thermo Fisher, Waltham, MS, USA). 50 μg of total protein was loaded on 10% denaturing polyacrylamide gels and transferred onto nitrocellulose membranes (GE Healthcare, Chicago, IL, USA). The membranes were blocked in TBS‐T, 5% (w/v) skimmed milk for 1 h and incubated with the primary antibodies directed against PD‐L1 (CAL10 clone, abcam, 1:1000 dilution in TBS‐T, 5% (w/v) bovine serum albumin and β‐actin (clone, Abcam, Cambridge, UK, 1:2000 dilution in TBS‐T, 5% (w/v) BSA) overnight at 4°C. For detection, a secondary antibody conjugated with horseradish peroxidase (HRP) and the Lumi‐Light substrate (Roche Applied Science, Basel, Switzerland) were applied. The signal was recorded with a LAS3000 camera system (Fuji LAS3000, Fuji GmbH, Düsseldorf) using the Image Reader LAS3000 software. The immunostaining signals were subsequently analysed using the ImageJ software (NIH, Bethesda, MD, USA). Quantification was done by setting the peak values of the control protein (β‐actin) for each loaded sample as “1”.
3
Epac1 and Epac2 Immunoblotting
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Immunoblotting was performed essentially as described elsewhere (Gausdal et al. 2004 ). The membranes were probed with either mouse monoclonal anti‐Epac1 or mouse monoclonal anti‐Epac2. Bands representing the proteins of interest were detected using alkaline phosphatase‐conjugated secondary antibodies and CDP‐Star substrate (Tropix, Bedford, MA, USA) and band intensity determined in a Fuji LAS3000 (Fuji Photo Film, Tokyo, Japan).
4
Quantitative Western Blot Analysis of APM
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For Western blot analysis, 50 μg protein/lane was separated in 10% SDS-PAGE gels, transferred onto nitrocellulose membranes (Schleicher & Schuell, Munich, Germany) and stained with Ponceau S as previously described.35 (link) Immune detection was performed with the following APM-specific primary antibodies (Ab): anti-TAP1 (ab13516, Abcam, Cambridge, United Kingdom), anti-TPN (ab13518, Abcam) and anti-TAP2, anti-MHC class I heavy chain (HC), anti-LMP2 and anti-LMP10, kindly provided by Soldano Ferrone. Staining with anti-GAPDH (Cell Signaling Technology) served as loading control. The membranes were then stained with suitable horseradish peroxidase (HRP)–conjugated secondary antibodies (DAKO, Hamburg, Germany or Cell Signaling Technology, Danvers, USA), before the signal was visualized with the Pierce Western Blot Signal Enhancer substrate (Thermo Scientific) and recorded with a LAS3000 camera system (Fuji LAS3000, Fuji GmbH, Düsseldorf) using the Image Reader LAS3000 software. The immunostaining signals were subsequently analyzed using the ImageJ software (NIH, Bethesda, MD). Relative protein expression levels are provided as arbitrary units by setting the peak values of the corresponding GAPDH signals to 1.
5
Quantification of Nitrotyrosine Protein Expression
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In total, 10 mg of MAT, or 4-6 branches of small mesenteric arteries (SMA) were homogenized in lysis buffer (Cellytic™ MT Mammalian Tissue Lysis/Extraction Reagent, Sigma-Aldrich, St. Louis, MO, United States). Protein concentration was assessed using BCA™ Protein Assay Kit (ThermoScientific, Rockford, IL, United States), and equal amounts of protein were separated by SDS-PAGE and transferred to PVDF membranes (Bio-Rad, Hercules, CA, United States). Protein expression was detected using anti-nitrotyrosine primary antibody (Abcam, Cat#ab7048, 1:500) anti-tubulin primary antibody (Abcam, Cat#ab6160, 1:5,000), Horseradish peroxidase-conjugated secondary antibodies were used. Signals were visualized by enhanced chemiluminescence (Santa Cruz Biotechnology, Santa Cruz, CA, United States), scanned densitometrically using Fuji LAS3000 and quantified with Multigauge software (Fujifilm). The relative amounts of protein expression were quantified to those of the corresponding m Leprdb control, which was set to a value of 1.0 (27 (link), 32 (link)).
6
Protein Profiling of Xenograft Samples
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Protein lysates were prepared from snap frozen xenografts, and proteins (400 μg) were incubated with the Rat Cytokine Array Kit, Panel A, and the Human Angiogenesis Array Kit (R&D Systems; Minneapolis, MN, USA) according to the manufacturer's instructions. Collection and quantification of pixel densities from membranes was performed using the Fuji Las3000 (Fujifilm; Tokyo, Japan) and Image Reader LAS-3000 (Version 2.2; Fujifilm). Arrays for all samples were performed in duplicate, and an average density was calculated for each corresponding protein probed on the arrays based on the two pixel densities determined for each experiment.
7
Immunoblotting with Chemiluminescence Detection
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Proteins separated by SDS-PAGE were transferred to nitrocellulose membranes and processed for immunoblotting using Super Signal West Pico Chemiluminescent substrate (Thermo Fisher Scientific). Images of immunoblots were captured with an imager (Fuji LAS-3000; Fujifilm) or with CL-XPosure film (Thermo Fisher Scientific) within the linear range and quantified by densitometry using the Analyze gels function in ImageJ.
8
Quantification of Mitochondrial DNA Content
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qPCR was used to amplify mtDNA and nucDNA as described before7 (link)15 . Briefly, the total DNA of cultured neurons was extracted and purified using the genomic DNA extraction kit (Qiagen Sciences, CA, USA). The total DNA derived from neurons in one well from 12-well plates was added to the polymerase chain reaction (PCR) mixture with GoTaq Flexi DNA Polymerase (Promega, WI, USA). The PCR products were separated by a 1% agarose gel and stained with ethidium bromide (0.5 mg/mL). The images were acquired by scanning with a Fuji LAS 3000 densitometer (Fuji, Tokyo, Japan). The densities of the mtDNA and nucDNA bands were calculated using ImageJ software. The density ratio of mtDNA to nucDNA was used to determine the relative mtDNA content and mitochondrial mass.
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