HCS-2/8 cells were treated with 5-HT, one of the agonists, or a combination of 5-HT and an antagonist. After 24 h, the cell lysates were prepared, and Western blot analysis was performed as described previously [13 ]. Briefly, proteins isolated from these cells were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and were then transferred to polyvinylidene difluoride (PVDF) membranes (Millipore) by using a semi-dry transfer apparatus (Atto Corp., Tokyo, Japan). Blots were then reacted overnight at 4°C with primary antibodies used at a predetermined dilution. Then, after washing with Tris-buffered saline-Tween 20 (TBST) and TBS buffers, the blots were incubated for 60 min at room temperature with secondary antibodies conjugated with horseradish peroxidase (HRP). Subsequently, the membranes were washed with TBST and TBS buffers, and the bands were detected with the chemiluminescence substrate by using a LAS-4000 mini image analyzer (Fuji Film, Tokyo, Japan). The band intensities were determined by using Multi Gauge ver. 3.0 soft (Fuji Film).
Las 4000 mini image analyzer
Manufactured by Fujifilm
Sourced in Japan
The LAS-4000 mini image analyzer is a compact and versatile tool for image capture and analysis. It features a high-resolution camera, automated image acquisition, and a suite of analysis tools to quantify various aspects of imaged samples. The core function of this product is to enable efficient and precise imaging and measurement of a wide range of biological and industrial samples.
Automatically generated - may contain errors
Lab products found in correlation
Multi gauge software, by Fujifilm (5 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Semi dry transfer apparatus, by ATTO Corporation (2 mentions)
M per lysis buffer, by Thermo Fisher Scientific (2 mentions)
Ecl western blotting detection reagent, by GE Healthcare (2 mentions)
β actin, by Cell Signaling Technology (2 mentions)
Radioimmunoprecipitation assay buffer, by Cell Signaling Technology (1 mentions)
Icam 1, by Abcam (1 mentions)
Ve cadherin, by Abcam (1 mentions)
Mg132, by Merck Group (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Merck Group (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Ecl prime western blotting detection kit, by GE Healthcare (1 mentions)
Immobilon p membrane, by Merck Group (1 mentions)
P stat3, by Cell Signaling Technology (1 mentions)
Interleukin 6 (il 6), by Abcam (1 mentions)
P akt, by Cell Signaling Technology (1 mentions)
Ripa buffer, by Cell Signaling Technology (1 mentions)
Chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Halt protease inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
15 protocols using las 4000 mini image analyzer
1
Western Blot Analysis of 5-HT Signaling
2
Immunoblotting Protocol for Protein Analysis
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Immunoblotting was performed as described previously [23 (link), 24 (link)]. In brief, cells were treated with the indicated drug and lysed. Samples were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes that were then blocked with 5% skimmed milk in TBS-Tween buffer (50 mM Tris-HCL [pH 7.4], 150 mM NaCl, and 0.05% Tween 20). Immunoblotting was carried out using primary antibodies (Table S2 ) appropriately diluted in TBS-Tween buffer containing 5% BSA and 0.05% sodium azide. Signals were detected with the appropriate horseradish peroxidase-conjugated secondary antibodies appropriately diluted in TBS-Tween buffer. Images were visualized with a LAS-4000 mini-image analyzer (FUJIFILM, Tokyo, Japan) and analyzed with MultiGauge software (FUJIFILM).
3
Western Blot Analysis of Protein Extracts
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Cells were collected and lysed with lysis buffer (50 mM Tris-HCl, pH 7.5, 1% [vol/vol] Triton X-100, 5% [vol/vol] glycerol, 5 mM EDTA, and 150 mM NaCl). After centrifugation at 18,000 × g for 15 min at 4°C, supernatants were collected. Threefold-concentrated Laemmli’s sample buffer (0.1875 M Tris-HCl, pH 6.8, 15% [vol/vol] β-mercaptoethanol, 6% [wt/vol] SDS 30% [vol/vol] glycerol, and 0.006% [wt/vol] bromophenol blue) was added to the supernatants, and the samples were boiled for 3 min. Proteins were separated by SDS–PAGE and transferred onto a nitrocellulose membrane (Advantec TOYO, Tokyo, Japan). The membrane was blocked with TBST (10 mM Tris-HCl, pH 7.4, 0.1 M NaCl, and 0.1% Tween-20) containing 5% (wt/vol) nonfat skim milk and probed with specific antibodies diluted with TBST containing 5% (wt/vol) nonfat skim milk or 5% (wt/vol) bovine serum albumin (BSA) overnight at 4°C. After probing with HRP-conjugated secondary antibodies, the bound antibodies were detected with Western Lightning Plus-ECL. Image acquisition was performed with an LAS 4000 mini image analyzer (Fujifilm, Tokyo, Japan), and image analysis was done by MultiGauge software (Fujifilm).
4
Protein Expression Analysis by Immunoblotting
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For immunoblotting, cells were lysed in M-PER lysis buffer (Thermo Scientific) containing aprotinin, sodium orthovanadate, and phenylmethylsulfonyl fluoride, and then subjected to SDS-polyacrylamide gel electrophoresis, protein transfer, and chemiluminescence using standard protocols. Blots were incubated with anti-PEG10 (1:1,000, 4C10A7, LSBio), anti-Smad1 (1:1,000, # 9743, CST), anti-p-Smad1/5/9 (1:1,000, D5B10, CST), anti-Smad2/3 (1:1,000, # 610842, BD Biosciences), anti-p-Smad3 (1:1,000, C25A9, CST), or anti-tubulin (1:1000, DM1A, Sigma) antibodies and then with horseradish peroxidase-conjugated anti-rabbit or anti-mouse secondary antibodies (1:10,000, CST). Chemiluminescent signals were detected using an LAS 4000 Mini Image Analyzer (Fujifilm). Band intensities of PEG10 and tubulin were quantified using ImageJ 1.50i software (National Institutes of Health, USA). The band intensity of PEG10 was normalised to that of tubulin.
5
Western Blot Analysis of Cell Signaling
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The cells were lysed and collected in radioimmunoprecipitation assay buffer (Cell Signaling Technology, Beverly, MA, USA). The total protein concentration was determined using the BCA Protein Assay Kit (Pierce, Rockford, IL, USA). An equal amount of proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. The membrane was blocked with 5% skim milk/TBST for 60 min. The antibodies used are specific for STAT3 (1:1,000, Cell Signaling Technology, 12640S, Beverly, MA, USA), pSTAT3 (1:1,000, Cell Signaling Technology, 9145S), VE-Cadherin (1:1,000, Abcam, ab33168, Cambridge, UK), ICAM-1 (1:2,000, Abcam, ab53013), and β-actin (1:5,000, Cell Signaling Technology, 4970), with anti-rabbit HRP-labeled antibody as the secondary antibody (1:5,000, Cell Signaling Technology, 7074). The signals were developed using ECL Western Blotting Detection Reagent (GE Healthcare, Little Chalfont, UK) and detected using LAS-4000 mini image analyzer (FUJIFILM, Tokyo, Japan). The levels of pSTAT3, VE-Cadherin and ICAM-1 were normalized to that of β-actin by densitometry using Image J.
6
Western Blot Protein Analysis
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Cells were collected and lysed with lysis buffer [50 mM Tris-HCl (pH 7.5), 1% (v/v) Triton X-100, 5% (v/v) glycerol, 5 mM EDTA and 150 mM NaCl]. After centrifugation at 18 000 g for 15 min at 4°C, supernatant was collected and boiled for 3 min with 3-fold concentrated Laemmli sample buffer [0.1875 M Tris-HCl (pH 6.8), 15% (v/v) β-mercaptoethanol, 6% (w/v) sodium dodecyl sulfate (SDS), 30% (v/v) glycerol and 0.006% (w/v) bromophenol blue]. Proteins were separated by SDS-polyacrylamide gel electrophoresis and then transferred onto a nitrocellulose membrane (Advantec TOYO; Tokyo, Japan). The membrane was blocked with Tris-buffered saline with Tween 20 (TBST) [10 mM Tris-HCl (pH 7.4), 0.1 M NaCl and 0.1% Tween-20] containing 5% (w/v) non-fat skim milk and probed with specific antibodies diluted in TBST containing 5% (w/v) non-fat skim milk, overnight at 4°C. After probing with HRP-conjugated secondary antibodies, the bound antibodies were detected with Western Lightning Plus-ECL. Images were captured using a LAS 4000 mini image analyzer (Fujifilm; Tokyo, Japan) and analyzed with MultiGauge software (Fujifilm).
7
Immunoblotting Analysis of RGA Protein
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Plant protein extracts of 2-week-old SD grown plants dissected for apices and leaves at ZT8 were prepared in extraction buffer (50 mM Tris-HCl, 150 mM NaCl, 0.5% Triton-X100, 10 μM MG132, 0.1 μM PMSF and Protease Inhibitor Cocktail, Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO, USA) and a total of 30 μg of plant protein extract was used for immunoblots. Western analysis was performed with anti-rabbit-RGA antibody (AGRISERA), and immunoblots were incubated with SuperSignal Femto West Substrate (Thermo Fisher Scientific) and detected with a LAS-4000 Mini-image analyzer (Fujifilm).
8
Protein Extraction and Immunoblotting from Arabidopsis
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Proteins were extracted from bud clusters or flowers from Arabidopsis inflorescences. Samples were frozen in liquid nitrogen, ground to a fine powder, suspended in SDS-PAGE sampling buffer (Laemmli, 1970) , heated at 94°C for 5 min, and centrifuged to remove insoluble materials. The extracted proteins were subjected to SDS-PAGE (Laemmli, 1970) , followed by transfer to Immobilon-P membranes (Millipore) as described by Towbin et al. (1979) . Primary antibodies were used at the following dilutions for immunoblotting: anti-ERdj3A, 1:1,000; anti-ERdj3B, 1:1,000; anti-P58 IPK , 1:1,000; anti-BiP, 1:5,000 (Yamamoto et al., 2008) ; and anti-actin, 1:2,000 (Affinity BioReagents). Horseradish peroxidase-labeled anti-rabbit IgG or anti-mouse IgG (GE Healthcare) was used as a secondary antibody at 1:5,000 dilution. Signals were detected using an ECL Prime western blotting detection kit (GE Healthcare) and an LAS4000 mini image analyzer (Fujifilm).
9
Immunoblotting Protein Detection Protocol
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Immunoblotting was performed as described previously [40 (link)]. In brief, cells were treated with the indicated drug and lysed. Samples were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinyldene difluoride membranes that were then blocked with 5% skimmed milk in TBS-Tween buffer (50 mM Tris-HCL [pH 7.4], 150 mM NaCl and 0.05% Tween 20). Immunoblotting was carried out using primary antibodies (Supplementary Table S3 ) and signals were detected with the appropriate horseradish peroxidase–conjugated second antibodies. Images were visualized with the LAS-4000 mini image analyzer (Fujifilm, Tokyo, Japan) and analyzed with MultiGauge software (Fujifilm).
10
Western Blot Analysis of Signaling Proteins
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The cells and EVs were lysed using radio immunoprecipitation assay (RIPA) buffer (Cell Signaling Technology). The total protein concentration was determined using a BCA Protein Assay kit (Pierce, Rockford, IL, USA). Equal amounts of whole protein extracts (10 μg for EVs and 20 μg for others) were separated on SDS-PAGE gels (10% for STAT3 and Akt and 12% for IL-6 analyses). Western blotting was conducted according to standard methods using antibodies specific for STAT3 (Cell Signaling Technology, 12640S, Beverly, MA), pSTAT3 (Cell Signaling Technology, 9145S), Akt (Cell Signaling Technology, 4685S), pAkt (Cell Signaling Technology, 4060S), IL-6 (Abcam, ab6672), β-actin (Cell Signaling Technology, 4970L), and a horseradish peroxidase (HRP)-conjugated secondary antibody as previously described20 (link). The signals were detected using ECL Western Blotting Detection Reagent (GE Healthcare, Little Chalfont, UK) and LAS-4000 Mini image analyzer (FUJIFILM, Tokyo, Japan).
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