For Western Blot analysis of IDE and β-actin protein levels, samples were prepared as described above, adjusted to equal protein amounts and loaded on 10–20% tris-tricine-gradient gels (Anamed Elektrophorese, Groß-Bieberau/Rodau, Germany). For measuring total Aβ degradation, the cell culture supernatant containing remaining human Aβ40 was also separated in tris-tricine-gradient gels. After transferring proteins onto nitrocellulose membranes, IDE, β-actin and human Aβ40 were detected with the primary antibodies ST1120 (1:2000), A5441 (1:5000) and W02 (1 μg/mL), all purchased from Merck, respectively. HRP-coupled antibodies W401 (anti-rabbit, 1:5000) (Promega, Mannheim, Germany) and P0260 (anti-mouse, 1:5000) (Dako, Hamburg, Germany) were utilized as secondary antibodies. Signal detection was performed with the enhanced chemiluminescence (ECL-) method (Perkin Elmer, Rodgau-Jügesheim, Germany) and densitometrical quantification of band intensity was carried out with Image Gauge software (Version 3.45) (Fujifilm). Total proteins were detected using Ponceau S staining as described in [52 (link)] before immunodetection.
A5441
Manufactured by Merck Group
Sourced in United States, United Kingdom, Germany, Spain, France, China
The A5441 is a laboratory instrument used for sample preparation and analysis. It provides precise and consistent sample handling capabilities. The core function of the A5441 is to enable efficient and reliable sample processing for a variety of scientific applications.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Merck Group (36 mentions)
Anti β actin, by Merck Group (13 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (13 mentions)
Pvdf membrane, by Merck Group (12 mentions)
Protease inhibitor cocktail, by Roche (10 mentions)
F1804, by Merck Group (10 mentions)
Bca kit, by Thermo Fisher Scientific (9 mentions)
Odyssey infrared imaging system, by LI COR (8 mentions)
A9044, by Merck Group (7 mentions)
Las 3000, by Fujifilm (6 mentions)
Chemidoc mp imaging system, by Bio-Rad (6 mentions)
Protease inhibitor cocktail, by Merck Group (6 mentions)
Immobilon p, by Merck Group (6 mentions)
Pvdf membrane, by Bio-Rad (6 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (6 mentions)
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (5 mentions)
Chemidoc xrs system, by Bio-Rad (5 mentions)
Ab125066, by Abcam (5 mentions)
P0448, by Agilent Technologies (5 mentions)
Ecl western blotting detection reagent, by GE Healthcare (5 mentions)
398 protocols using a5441
1
Quantification of IDE and Aβ Levels
2
Quantifying C9orf72 and SUMO2/3 Protein Levels
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Cell pellets obtained from cultured fibroblasts were homogenized with RIPA buffer (SDS 0.1%, NP40 1% and sodium deoxycholate 0.5% in PBS) containing protease inhibitors (cOmplete™, Mini, EDTA-free Protease Inhibitor Cocktail 4693159001, Roche, Indianapolis, IN, USA). Cell lysates were subjected to SDS-PAGE and electroblotted. Proteins were visualized by immunostaining with antibodies against C9orf72 1:5000 (Abcam, Marid, Spain, ab221137) SUMO2/3 1:1000 (Abcam, Marid, Spain, ab3742) and β-actin 1:5000 (A5441, Merck KGaA, Darmstadt, Alemania). Colorimetric detection (1708235 Opti-4CNTM Substrate Kit, Bio-Rad, Hercules, CA, USA) and ImageJ software were used for the densitometry analysis of protein expression.
3
Western Blot Analysis of Lumbar DRG Proteins
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Lumbar DRG were dissected from individual rats (vehicle: n = 6 rats, pre-CIPN: n = 5 rats) following standard procedures, in brief: rats were sacrificed, the dorsal part of the rib cage and the spinal column were removed, and the spinal column was carefully cut open at the dorsal side. The spinal cord was carefully pushed to the side and lumbar DRG (between L6–L3 spinal levels, i.e., 6 DRG/rat) were pulled out from their grooves using fine forceps. DRG were immediately frozen in liquid nitrogen until further use. Total protein was obtained by homogenization in RIPA lysis buffer containing protease and phosphatase inhibitors. 30 µg samples were separated on a 10% polyacrylamide gel by SDS-PAGE and transferred to PDVF membrane (0.45 µm, Millipore). Blots were blocked and incubated overnight at 4°C in rabbit anti – β-catenin (1:500, ab32572, Abcam, United Kingdom) and mouse anti –β-actin (1:5000, A5441, Merck, United Kingdom). The following day the membrane was incubated for 1 h at room temperature in goat anti-mouse IRDye® 680 (1:5000, 926-68070, LI-COR) and goat anti-rabbit Dylight 800 (1:5000, A23920-1, Abbkine) and scanned with the Odyssey Infrared Imaging System (LI-COR Biosciences). Densiometric analysis was performed using ImageJ (version 1.52p) and by normalizing expression to β-actin.
4
Quantitative Analysis of H3K9me3 in Cumulus Cells
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Cumulus cells grown in a 24-well were suspended in the SDS sample buffer (196-11022; Wako), and the lysed samples were subjected to SDS–PAGE (15% poly-acrylamide gel). The gels were transferred onto polyvinylidene difluoride membranes, and the membranes were blocked with 3% BSA in PBS-Tween (PBS-T) solution for 1 h. The blocked membranes were incubated with anti-H3K9me3 (ab8898; Abcam) or anti-ACTB antibodies (A5441; Merck) overnight at 4°C. After washing, the membranes were incubated with HRP-conjugated donkey anti-rabbit IgG secondary antibody (AP182P; Millipore) or IRDye 680RD goat anti-rabbit IgG secondary antibody (925-68071; LI-COR), and visualized using ECL prime Western blotting detection reagents (RPN2232; Amersham) or Odyssey imaging system (LI-COR).
5
Analyzing Tight Junction Proteins
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Protein extraction and Western blot analysis were performed according to standard procedures using antibodies against claudin-3 (1:150, 34–1700, Thermo Fisher Scientific, Waltham, MA, USA), claudin-5 (1:250, sc-28670, Santa Cruz Biotechnology, Dallas, TX, USA), occludin (1:250, 611090, BD Biosciences, San Jose, CA, USA), ICAM-1 (1:250, sc1511, Santa Cruz Biotechnology, Dallas, TX, USA) VCAM-1 (1:2000, ab134047, Abcam, Cambridge, UK) and actin (1:500,000, A5441, Merck, Darmstadt, Germany) [34 (link)].
6
Western Blotting of PTEN, hTERT, and PI3K
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Western blotting was performed as previously described [37] (link) using the following antibodies: anti-PTEN mouse monoclonal (ab130224, Abcam, MA,USA), anti-hTERT rabbit polyclonal (ab130224, Abcam, MA, USA), anti-p-AKT rabbit polyclonal (ab81283, Abcam, MA, USA), anti-PI3K mouse polyclonal (ab86714, Abcam, MA, USA) and anti-β-actin mouse monoclonal (A5441, Sigma-Aldrich, MO, USA). Signals were observed using an Odyssey Infrared Imaging System (LI-COR Biosciences, NE, USA).
7
Quantifying Protein Expression via Western Blot
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Cells were resuspended in lysis buffer (50 mM Tris-HCl pH 7.4, 130 mM NaCl, 1 mM EDTA, 1% Triton X-100) containing protease inhibitors (Complete, Roche Applied Science, Indianapolis) and protein concentration was measured using the Bradford assay (#5000006, BioRad, Hercules, CA, USA). Protein samples (30 μg/lane) were subjected to SDS-PAGE and then transferred to PVDF membranes (Immobilon-PSQ PVDF Membrane, Merck Millipore, Darmstadt, Germany). After blocking, membranes were incubated with primary antibodies against the following proteins: α-tubulin (1:5000, T6199, Sigma-Aldrich, St. Louis, MO, USA), acetylated α-tubulin (1:1000, T7451-25UL, Sigma-Aldrich, St. Louis, MO, USA), and β-actin (1:10,000, A5441, Sigma-Aldrich, St. Louis, MO, USA). β-actin was used as the loading control. Goat Anti-Mouse IgG (H+L) DryLightTM 680 Conjugated (1:10,000, 35518, Invitrogen, Waltham, MA, USA) was used as the secondary antibody. Immunoblots were incubated for 1 h at room temperature and developed using Odyssey infrared imaging system (LI-COR Biosciences, Lincoln, NE, USA). Protein expression levels were calculated using ImageJ Software (ImageJ2, version 2.3.0/1.53q).
8
SLC7A11 Antibody Specificity Validation
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SLC7A11 primary antibody specificity (Rabbit monoclonal, Ab37185, Abcam, UK) was determined using Western blotting in cell line lysates: HCC1500, ZR-751, MDA-MB-436, MCF7 and T47D (American Type Culture Collection; Rockville, MD, USA at a dilution of 1:2000. Donkey anti-rabbit (1:15,000, IRDye680 CW, 926–32213, LI-COR Bioscience) was used as a fluorescent secondary antibody. Mouse monoclonal anti-β-actin primary antibody (1:5,000, A5441, Sigma-Aldrich) with donkey anti-mouse fluorescent secondary (1:15,000, IRDye 800CW, 926–68072, LI-COR Bioscience) was used as a control. The Odyssey Fc machine (LI-COR Bioscience) was used to visualize blots showing specific bands at the predicted size of approximately 55 KDa (Supplementary Figure S1).
9
Western Blot Analysis of Liver Proteins
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Total protein extracts from normal tissues, HCC liver tissues and cell lysates were obtained by RIPA buffer treatment and examined via western blotting as described previously [16 (link)]. Blots were imaged using ChemiDoc imaging system (Bio-rad, Hercules, CA, USA). Serum proteins were diluted 50 times before being subjected to western blotting. Antibodies against p65 (1:1000, 8242, CST, Danvers, MA, USA), p-p65 (1:1000, 3033, CST), PCNA (1:2000, 13,110, CST), ARRB1 (1:1000, 12,697, CST), TNF-α (1:1000, 6945, CST), PI3K (1:1000, 4249, CST), Akt (1:1000, 9272, CST), p-Akt (1:1000, 4060, CST), GSK3β (1:1000, 12,456, CST), p-GSK3β (1:1000, 9323, CST), mTOR (1:1000, 2983, CST), p-mTOR (1:1000, 5536, CST) and β-actin (1:3000, A5441, Sigma-Aldrich, St Louis, MO, USA) were used as primary antibodies. Goat anti-mouse (1:5000, 7076, CST) or goat anti-rabbit (1:5000, 7074, CST) HRP-linked antibodies were used as secondary antibodies. After quantifying the densitometry of each target protein and β-actin, the results were expressed as normalized ratios.
10
Western Blot Analysis of ABCA1
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iRPE cells were thawed at p3, seeded on Matrigel-coated plates at 1.5 × 105 cells/cm2 and cultivated in FMN medium. One day before harvesting, cells were treated with 1 μM LXR agonist (T090137, Sigma-Aldrich, St. Louis, MO, USA) or DMSO overnight for 16 h. Cell lysates were prepared by direct lysis in RIPA buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% Triton X-100, Protease Inhibitor Cocktail tablet (Roche, Basel, Switzerland)) and sonication. Protein concentration was determined using a BCA Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). Then, 50 μg protein lysates were separated by SDS-PAGE and transferred onto nitrocellulose membranes. Membranes were blocked in 5% non-fat milk in TBS-T and incubated with primary antibodies overnight at 4 °C: rabbit anti-ABCA1 (1:250, NB400-105, Novus Biologicals, Littleton, CO, USA), mouse anti-ACTB (1:10,000, A5441, Sigma-Aldrich, St. Louis, MO, USA). After washing, membranes were incubated with HRP-conjugated secondary antibodies at RT for 1 h and the signal developed using enhanced chemiluminescence (ECL) substrate (PerkinElmer, Schwerzenbach, Switzerland) and an X-ray developer. Band intensities were quantified using ImageJ and normalized to actin levels.
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