The cultured cells were lysed with Pierce RIPA Buffer (Thermo Scientific, Waltham, MA, USA) with Halt protease inhibitor Cocktail (Thermo Scientific). Lysates mixed with sample buffer were electrophoretically separated and transferred onto membranes. Membranes were blocked with 5% skim milk, followed by incubations with anti-human E-cadherin antibody (24E10; Cell Signaling Technology, Danvers, MA, USA), anti-human N-cadherin antibody (#610920; BD Biosciences, San Jose, CA, USA), anti-human vimentin antibody (D21H3; Cell Signaling Technology), anti-human Smad2/3 (#07-408; Millipore, Billerica, MA, USA), anti-human phospho-Smad2 (Ser465/467) (#AB3849; Millipore), anti-human phospho-Smad3 (Ser423/425) (#07-1389; Millipore), and anti-human β-actin antibody (#4963; Cell Signaling Technology). After washing with TBS-0.05% Tween, membranes were incubated with HRP-conjugated anti-mouse or anti-rabbit IgG. After washing with TBS-0.05% Tween, membranes were incubated with ECL Prime Western Blotting Detection Reagent (GE Healthcare, Little Chalfont, UK). Signals were detected and analysed using a Luminescent Image Analyzer LAS-4000 (Fuji Film, Tokyo, Japan). Signals were detected and analysed using a Luminescent Image Analyzer LAS-4000 (Fuji Film).
Las 4000 luminescent image analyzer
Manufactured by Fujifilm
Sourced in Japan, United States, United Kingdom, Argentina
The LAS-4000 is a luminescent image analyzer manufactured by Fujifilm. It is designed to capture and analyze luminescent images, such as those produced by chemiluminescent or bioluminescent samples. The device features a high-sensitivity camera, advanced imaging software, and various imaging modes to support a wide range of applications in life science research and other fields.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Thermo Fisher Scientific (8 mentions)
Ripa buffer, by Thermo Fisher Scientific (6 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (6 mentions)
Protease inhibitor cocktail, by Roche (5 mentions)
Pvdf membrane, by Merck Group (5 mentions)
Luminata crescendo western hrp substrate, by Merck Group (4 mentions)
Immobilon western chemiluminescent hrp substrate, by Merck Group (4 mentions)
Bca protein assay kit, by Beyotime (4 mentions)
Polyvinylidene fluoride membrane, by Merck Group (4 mentions)
β actin, by Merck Group (4 mentions)
Ecl kit, by Thermo Fisher Scientific (4 mentions)
α mcherry, by Abcam (3 mentions)
Clarity western ecl substrate kit, by Bio-Rad (3 mentions)
Ripa buffer, by Merck Group (3 mentions)
Protease inhibitor cocktail, by Merck Group (3 mentions)
Nupage bis tris gel, by Thermo Fisher Scientific (3 mentions)
Polyvinylidene difluoride membrane, by Merck Group (3 mentions)
Enhanced chemiluminescence system, by Cytiva (2 mentions)
Bradford assay, by Bio-Rad (2 mentions)
Ecl prime western blotting detection regent, by GE Healthcare (2 mentions)
143 protocols using las 4000 luminescent image analyzer
1
Western Blot Analysis of Epithelial-Mesenchymal Transition Markers
2
Western Blot Analysis of Cellular Proteins
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Western blot was performed using an enhanced chemiluminescence system [22 (link),23 (link),29 (link),30 (link),31 (link)]. Briefly, equal amounts of extracted cellular proteins were separated by 10% SDS–polyacrylamide gels and electrotransferred onto polyvinylidene difluoride membranes. After blocking with 3% BSA in PBS, the membranes were incubated with primary antibody. After washing with PBS-0.1% Tween 20, filters were probed with horseradish peroxidase–conjugated sheep anti-rabbit IgG or rabbit anti-mouse IgG (Cell Signaling; Beverly, MA, USA). Immunoreactivity was detected by an enhanced chemiluminescence system (Amersham Biosciences, Buckinghamshire, UK). The chemiluminescent signal was captured with a Fujifilm luminescent image LAS-4000 analyzer (Fujifilm, Tokyo, Japan). Densitometric quantitation of the blots was performed using the Scion Image software (Scion Corporation).
3
Western Blot Analysis of Ubap1 Protein
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Western blotting was performed using an enhanced chemiluminescence system. Immunoblots were obtained for total protein extracts of the brains of 7-month-old wild-type (N = 3) and heterozygous Ubap1+/E176Efx23 knock-in mice (N = 3). Briefly, the proteins were harvested from brain tissue by homogenization in RIPA buffer with a protease inhibitor cocktail (Roche). Equal amounts of proteins were separated on 10% SDS–polyacrylamide gels and then electrotransferred onto polyvinylidene difluoride membranes. After blocking with 3% BSA in PBS, the membranes were incubated with a primary antibody. After washing with PBS-0.1% Tween 20, filters were probed with horseradish peroxidase-conjugated sheep anti-rabbit IgG or rabbit anti-mouse IgG (Cell Signaling; Beverly, MA, USA). Immunoreactivity was detected with an enhanced chemiluminescence system (Amersham Biosciences, Buckinghamshire, UK). The chemiluminescent signals were captured with a Fujifilm luminescent image LAS-4000 analyzer (Fujifilm, Tokyo, Japan). Two kinds of rabbit polyclonal anti-UBAP1 antibodies (Sigma-Aldrich, SAB1307218 and Invitrogen, PA5-49644) were used in this study.
4
Viral Protein Detection in Infected Cells
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At 24 h p.i., virus-infected cells were washed with PBS and lysed using M-PER buffer (Thermo Fisher Scientific, Waltham, MA, USA) containing 0.5% protease inhibitor cocktail (Pierce, Rockford, IL, USA). The cell lysates were clarified by centrifugation, and the total protein content was determined by the Bradford assay (Bio-Rad, Hercules, CA, USA). Equal amounts of protein were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and electro-transferred to a PVDF membrane. Viral proteins were detected using primary antibodies specific for DENV NS3, DENV E, DENV prM, ZIKV E, and ZIKV prM followed by a horseradish peroxidase (HRP)-conjugated goat anti-mouse or anti-rabbit secondary antibody. As a loading control, cellular β-actin was detected with an anti-β-actin-specific primary antibody and HRP-conjugated goat anti-mouse secondary antibody. After the addition of a chemiluminescent HRP substrate (SuperSignal West Pico Chemiluminescent Substrate; Pierce), images were obtained using a LAS-4000 Luminescent Image Analyzer (Fujifilm, Tokyo, Japan).
5
Protein Expression Analysis in Mouse Tissues
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Mouse-tissue protein extracts were subjected to immunoblot analysis with specific antibodies against phospho-AMPK, AMPK, PPARα, C/EBPα (Santa Cruz Biotechnology, Santa Cruz, CA, USA), FABP4 (Cell Signaling Technology, Danvers, MA, USA), and GAPDH (Bioss Antibodies, Boston, MA, USA) for the analysis of phosphorylated or total protein expression. GAPDH served as a loading control. Tissue proteins were fractionated by electrophoresis on 10% or 12% SDS-PAGE gels and transferred onto polyvinylidene fluoride membranes. The membranes were blotted with antibodies specific for each protein. Protein expression was determined using an Immobilon Western Chemiluminescent HRP Substrate (Merck Millipore, Burlington, MA, USA) and LAS-4000 Luminescent-Image Analyzer (Fuji Photo Film, Tokyo, Japan). The intensities of the detected bands were quantified using Fujifilm Image MultiGauge (Fuji Photo Film, Tokyo, Japan).
6
Quantifying Moesin Expression in Transgenic Mouse Brains
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Total proteins were extracted from wild-type and Human BAC2 Tg mouse brains and separated by 10% polyacrylamide gel electrophoresis. Proteins were transferred to PVDF membrane (Millipore IPVH00010) with AE-6687 HorizeBLOT system (ATTO). Primary antibodies and dilutions used were rabbit anti-Moesin (Msn, 1:1000, Cell Signaling 3146) and rabbit anti-GAPDH (14C10, 1:2000, Cell Signaling 2118). Secondary antibody was anti-rabbit IgG, horseradish peroxidase-linked (GE Healthcare NA9340V) diluted 1:5000. Signal bands were detected by using ECL Prime Western Blotting Detection Regent (GE Healthcare RPN2232) and images were captured on LAS-4000 Luminescent Image Analyzer (Fujifilm). Total density of each Msn and Gapdh protein band was determined with ImageJ (version: 2.0.0) software. For each sample, the ratio of Msn to Gapdh total density was calculated. Msn-to-Gapdh ratios were analyzed by Student’s t test for comparison of wild-type to Human BAC2 Tg brain samples (n = 3 for each brain regions).
7
Immunoprecipitation and Immunoblotting Protocol
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Immunoprecipitation and immunoblotting were performed as previously described [15] (link). Briefly, cells were harvested after washing with ice-cold phosphate buffered saline and lysed in an ice-cold lysis buffer. Immunoprecipitation was performed by incubating cell lysates with an appropriate primary antibody for 16 hours at 4°C. Immunoprecipitated complexes were then pulled down using protein G-Sepharose beads (GE Healthcare). Cell lysates or immunoprecipitated proteins were resolved by electrophoresis and transferred to PVDF membranes. Proteins were visualized using appropriated primary antibody followed by horseradish peroxidase–conjugated secondary antibody. Blots were developed with the enhanced chemiluminescence solution. Images were obtained and analyzed by a LAS4000 luminescent image analyzer (Fuji Photo Film). All results are representative of at least three independent experiments.
The study protocol was reviewed and approved by the Institutional Review Board of Chonnam National University Medical School Research Institution (2013-19).
The study protocol was reviewed and approved by the Institutional Review Board of Chonnam National University Medical School Research Institution (2013-19).
8
Immunoblot Analysis of Protein Targets
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Immunoblots were carried out as described71 . Briefly, rabbit polyclonal α-FLAG (Rockland, dilution 1:2000), α-mCherry (Biovision, dilution 1:10000), α-PilC74 (link) (dilution 1:5000) antibodies were used together with horseradish-conjugated goat anti-rabbit immunoglobulin G (Sigma-Aldrich, dilution 1:10000) as secondary antibody. Blots were developed using Luminata Crescendo Western HRP Substrate (Millipore) and visualized using a LAS-4000 luminescent image analyzer (Fujifilm).
9
Microscopic Lung Imaging and Western Blot Analysis
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Lung sections were documented with a DM5000 microscope (Leica Camera, Wetzlar, Germany) equipped with a Leica DFC300FX digital camera. Images were processed and analyzed with Adobe Photoshop CS5 (Adobe, San Jose, CA, USA) and ImageJ software (https://imagej.nih.gov ). Western blots Blots were documented on a LAS-4000 luminescent Image Analyzer (Fuji, Tokyo, Japan).
10
Western Blotting Analysis of Intestinal Proteins
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Western blotting analysis was conducted as described by Xu et al. [29 (link)], with some modifications. Briefly, intestinal tissues were homogenized, and protein concentrations
were determined with the BCA Protein Assay Kit (Beyotime, Jiangsu, China). Then, protein from each sample (20 µg) was separated by SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. The
membranes were blocked with 5% skimmed milk in blocking buffer containing 0.1% TBST for 1.5 h at room temperature, and then incubated with primary antibodies against p62 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), mTOR, and
β-actin (Cell Signaling Technology, Danvers, MA, USA) for 12 h at 4°C. After three consecutive washes with TBST, the membranes were incubated with the secondary antibody (horseradish peroxidase-conjugated goat anti-rabbit
immunoglobulin G; Cell Signaling Technology). Antibody-bound protein bands were detected using enhanced chemiluminescence reagents (ECL-Kit; Beyotime) followed by autoradiography. The blots were scanned using a LAS-4000
Luminescent Image Analyzer (Fuji Film, Tokyo, Japan), and the antigen-antibody complexes were quantified with Quantity One software (Bio-Rad Laboratories, Hercules, CA, USA).
were determined with the BCA Protein Assay Kit (Beyotime, Jiangsu, China). Then, protein from each sample (20 µg) was separated by SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. The
membranes were blocked with 5% skimmed milk in blocking buffer containing 0.1% TBST for 1.5 h at room temperature, and then incubated with primary antibodies against p62 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), mTOR, and
β-actin (Cell Signaling Technology, Danvers, MA, USA) for 12 h at 4°C. After three consecutive washes with TBST, the membranes were incubated with the secondary antibody (horseradish peroxidase-conjugated goat anti-rabbit
immunoglobulin G; Cell Signaling Technology). Antibody-bound protein bands were detected using enhanced chemiluminescence reagents (ECL-Kit; Beyotime) followed by autoradiography. The blots were scanned using a LAS-4000
Luminescent Image Analyzer (Fuji Film, Tokyo, Japan), and the antigen-antibody complexes were quantified with Quantity One software (Bio-Rad Laboratories, Hercules, CA, USA).
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