Immunoblotting analyses were carried out as described previously24 (link). Digital images were captured using the LAS-1000 Plus (Fujifilm). The following primary antibodies were used: mouse anti-Flag M2 (Sigma), anti-Myc 9E10 (Santa Cruz), and anti-β-actin 2F3 (Wako); rabbit anti-Myc and anti-SP-B (Santa Cruz), anti-CtBP1, anti-CtBP2, anti-SP-C, anti-Muc1 and anti-Abca3 (Abcam), anti-Foxp1 (CST), anti-Foxp2 (Sigma), anti-phospho-ERK1/2 (CST), and anti-ERK2 (Santa Cruz). An affinity-purified anti-MCRIP1 antibody was made in-house as described previously24 (link). All antibodies were used at a dilution of 1:1000 for western blotting. Full size western blot images are shown in Supplementary Fig. 6 .
Las 1000plus
Manufactured by Fujifilm
Sourced in Japan
The LAS-1000plus is a laboratory equipment product offered by Fujifilm. It is a device used for image capture and analysis. The core function of the LAS-1000plus is to provide high-quality image documentation and quantification for various applications in scientific research and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Quantity one 1 d analysis software, by Bio-Rad (4 mentions)
Bovine serum albumin (bsa), by Merck Group (4 mentions)
Anti myc 9e10, by Santa Cruz Biotechnology (2 mentions)
Ecl western blotting detection reagent, by Merck Group (2 mentions)
Anti gad67, by Merck Group (2 mentions)
Anti βiii tubulin, by Merck Group (2 mentions)
4 laemmli sample loading buffer, by Bio-Rad (2 mentions)
Flamingo stain, by Bio-Rad (2 mentions)
Immobilon p, by Merck Group (2 mentions)
Precision plus protein dual color standard, by Bio-Rad (2 mentions)
Anti flag m2, by Merck Group (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Bicinchoninic acid assay, by Thermo Fisher Scientific (2 mentions)
Mouse anti flag m2, by Merck Group (1 mentions)
Anti sp c, by Abcam (1 mentions)
Anti β actin 2f3, by Fujifilm (1 mentions)
Anti ctbp1, by Abcam (1 mentions)
Anti ctbp2, by Abcam (1 mentions)
Anti foxp2, by Merck Group (1 mentions)
Anti erk2, by Santa Cruz Biotechnology (1 mentions)
30 protocols using las 1000plus
1
Western Blot Immunoanalysis Protocol
2
Western Blot Analysis of Hippocampal Proteins
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Mouse hippocampus were prepared from P30 control and CCK-Cre;Erbb4F/F homogenized in lysis buffer containing 50 mM Tris pH 7.5, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 0.5 mM prevanadate with Protease Inhibitor Cocktail (Complete, Mini, Roche). Samples were denatured and run on 10% SDS–PAGE gels. Gels were electrophoretically transferred onto PVDF membranes (Whatman GmbH). Membranes were blocked with 5% BSA (Sigma) in TBS (20 mM Tris-HCl, pH 7.5, 150 mM NaCl) for 1 h and probed with primary antibodies: anti-β III-tubulin (1:2000; Sigma #T8660), anti-GAD65 (1:1000; Millipore #MAB351R) and anti-GAD67 (1:1000; Chemicon #MAB5406), in 1% BSA in TBS + 0.1% Tween20. Subsequently, they were treated with horseradish-peroxidase-conjugated secondary antibodies and ECL western blotting detection reagents (Immobilon, Millipore) Signals were acquired as 16 bit images with a luminescent image analyzer (LAS-1000PLUS; Fujifilm) and quantified with Quantity One 1D Analysis Software (Bio-Rad Laboratories).
3
Quantifying SLC37A2 Protein Levels in AoSMCs
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Total protein was collected from AoSMC samples cultured under the same conditions used for real-time PCR analysis. These samples were lysed using Tris-buffered saline (pH 7.5) supplemented with 5% NP-40 and a 1% protease inhibitor cocktail. The protein concentration samples was measured using the BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA), and the concentration was adjusted. For western blotting, 10 µg of protein was separated in a 10% polyacrylamide gel using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The membrane was treated with SLC37A2 antibody (Novus Biologicals, Centennial, CO) or GAPDH antibody (GeneTex, Irvine, CA) as the primary antibodies, incubated with Rabbit IgG Horseradish peroxidase-conjugated antibody (R&D Systems, Minneapolis, MN) as the secondary antibody, and visualized using Amersham ECL Select (GE Healthcare, Buckinghamshire, England) and luminescent image analyzer (LAS-1000 plus, FUJIFILM, Tokyo, Japan). The band was quantified using ImageJ.
4
Isolation and Characterization of BV2 Exosomes
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The pellet from either BV2 cells or BV2-derived exosomes was lysated in PRO-PREP™ protein extraction solution (iNtRon Biotechnology, Seongnam, Korea), and the protein was loaded on 10% SDS polyacrylamide gel stained with Coomassie brilliant blue. Also, the protein was transferred to PVDF membranes for 2 h. Western blotting was confirmed with Alix (Cell signaling Technology, Beverly, MA, USA) antibody as an exosome marker. HRP-conjugated goat anti-mouse IgG (H + L) (Bethyl Laboratories, Montgomery, TX, USA) antibody was used as a secondary antibody, and the immune-reactive band was visualized using a luminescent image analyzer LAS-1000 plus (Fuji Photo Film, Tokyo, Japan). Transmission electron microscopy was performed to examine the size and morphology of the exosomes. Exosomes (6.40 µg/µL) isolated from BV2 cells were prepared on a carbon-coating grid and negatively stained using 2% uranyl acetate for 1 min45 (link). The prepared samples were observed using a JEOL 1200-EX II (Tokyo, Japan) equipped with an ES1000W Erlangshen CCD camera (Gatan, Pleasanton, CA, USA) at an accelerating voltage of 80 kV.
5
Immunoblotting and Immunoprecipitation Protocol
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Immunoblotting and immunoprecipitation were performed essentially as described previously39 (link). To analyze TM2D3 by immunoblotting, samples were denatured at 50 °C for 5 min before loading to the gels. The first antibodies on the membrane filters were visualized with secondary antibodies conjugated with horseradish peroxidase, a Western Lightning ECL Pro (PerkinElmer) chemiluminescent substrate, and a LAS-1000plus (Fujifilm) or ChemiDoc Touch (Bio-Rad) luminescent image analyzer; or secondary antibodies conjugated with near infrared dyes and Odyssey CLx (LI-COR).
6
Western Blot Analysis of Cell Signaling
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TS and AGTS cells were washed once with PBS and lysed on ice in 10 ml of buffer comprised of 62.5 mM Tris-HCl
(pH 6.8), 2% SDS, 10% glycerol, 2.5% β-mercaptoethanol and 1.25% bromophenol blue. The cells were then boiled
at 95 C for 5 min.
Electrophoresis was carried out on a 10% polyacrylamide gel, and separated proteins were transferred to a
nitrocellulose membrane (Hybond ECL, GE Healthcare, Piscataway, NJ, USA). The membrane was blocked in 5% skim
milk in washing buffer (25 mM Tris-HCl, 150 mM NaCl and 0.05% Tween-20) for 1 h and incubated with primary
antibodies (anti-rabbit P57/KIP2 polyclonal antibody [1:1000; Abcam, Cambridge, UK], anti-CHK1 mouse
monoclonal antibody [Santa Cruz] and anti-human G3PDH antibody [Trevigen, Gaithersburg, MD, USA]) overnight at
4 C, followed by anti-rabbit or anti-mouse horseradish peroxidase (HRP)-conjugated secondary antibodies
(1:1000; GE Healthcare). Signals were detected with an ECL Prime Western Blotting Detection Reagent (GE
Healthcare) and visualized by a luminescent image analyzer (LAS-1000plus; Fujifilm, Tokyo, Japan). Protein
samples were from 1 x 104 cells for P57/KIP2 detection and 20 μg proteins for CHK1 detection.
(pH 6.8), 2% SDS, 10% glycerol, 2.5% β-mercaptoethanol and 1.25% bromophenol blue. The cells were then boiled
at 95 C for 5 min.
Electrophoresis was carried out on a 10% polyacrylamide gel, and separated proteins were transferred to a
nitrocellulose membrane (Hybond ECL, GE Healthcare, Piscataway, NJ, USA). The membrane was blocked in 5% skim
milk in washing buffer (25 mM Tris-HCl, 150 mM NaCl and 0.05% Tween-20) for 1 h and incubated with primary
antibodies (anti-rabbit P57/KIP2 polyclonal antibody [1:1000; Abcam, Cambridge, UK], anti-CHK1 mouse
monoclonal antibody [Santa Cruz] and anti-human G3PDH antibody [Trevigen, Gaithersburg, MD, USA]) overnight at
4 C, followed by anti-rabbit or anti-mouse horseradish peroxidase (HRP)-conjugated secondary antibodies
(1:1000; GE Healthcare). Signals were detected with an ECL Prime Western Blotting Detection Reagent (GE
Healthcare) and visualized by a luminescent image analyzer (LAS-1000plus; Fujifilm, Tokyo, Japan). Protein
samples were from 1 x 104 cells for P57/KIP2 detection and 20 μg proteins for CHK1 detection.
7
Analysis of quinone-modified proteins
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After incubating the enzyme with the test chemical, 10-mM dithiothreitol and × 4 Laemmli sample loading buffer (Bio-Rad, CA, USA) were added to the reaction mixture. The sample (60-ng protein/lane) and Precision Plus Protein Dual Color Standard (Bio-Rad) were then applied to an SDS-polyacrylamide gel (SDS-PAGE, 10% acrylamide) and then migrated by electrophoresis. The proteins in gels were blotted onto a PVDF membrane (Immobilon-P, Merck), and quinones on the membrane were stained using NBT (0.24 mM) in 2-M glycine-NaOH (pH 10) as described previously [19 (link),20 (link)]. The same samples were separated using a gel, and the proteins in the gel were stained using Flamingo Stain (Bio-Rad) according to the manufactures’ protocol. Detection was performed using a LAS1000 plus (Fujifilm, Tokyo, Japan). This experiment was performed twice.
8
Quantitative Analysis of Recombinant RBP4
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Predetermined recombinant RBP4 proteins expressed by HEK293 cells were resolved on a polyacrylamide gel in parallel with five serum samples whose concentrations were determined by the IgA-based ELISA. After probing the Western blot filters with a rabbit anti-RBP4, phospho-image analysis was conducted using an image analyzer (LAS-1000Plus, FUJIFILM, Japan). Quantitation is represented in AU/mm2 according to the manufacturer’s instructions.
9
Western Blot Protein Detection
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Cells were lysed in Laemmli sample buffer (125 mM Tris, pH 6.7, 20% glycerol, 140 mM SDS [Sigma-Aldrich, G5516 and L3771]) supplemented with 3x complete protease inhibitor cocktail (Roche, 11697498001) and 40 µg/ml pepstatin A (Roche, 10359053001) at 10000 cells/µl. After addition of 0.1 M dithiothreitol and bromophenol blue (Sigma-Aldrich, D9779 and B0126), boiling and separation by 4–20% gradient SDS-PAGE, proteins were transferred onto polyvinylidene difluoride (PVDF) membranes using Bio-Rad Trans-Blot Turbo system. Membranes were blocked with DPBS containing 5% milk and 0.1% Tween-20 (Sigma-Aldrich, 274348) and labeled with the antibodies listed above. The signal was detected with Clarity Western ECL Substrate (Bio-Rad, 170–5061), and Luminescent Image Reader (LAS-1000Plus, Fujifilm, Tokyo, Japan), and quantified by densitometry with ImageJ software.
10
Western Blot Analysis of Protein Expression
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For the detection of proteins, the nitrocellulose membranes were blocked with 2% ECL blocking agent (GE) for 1 hour at R.T. For the detection of O-GlcNAcylation, membranes were blocked with 3% BSA for 1 hour at R.T. After incubation of the membranes with the appropriate primary antibodies overnight at 4°C, antibody-bound proteins on the membranes were visualized by using enhanced chemiluminescence (ECL prime, GE). An aliquot of HEK293 cell lysates containing 30 μg protein was used for the detection of endogenous AGFG1 and Nup62. Digitized images were captured by LAS-1000 Plus with a CCD camera (Fujifilm), and protein bands were quantified by densitometric analysis using Image gauge (Fujifilm). The detection of O-GlcNAcylation using succinylated WGA-HRP was carried out as described previously [16 (link)].
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