2 × 106 cells were seeded in a 10 cm dish and incubated as indicated. Cells were washed in PBS and lysed in 100 μl PLB. Clarified lysates were analysed by 15% SDS-PAGE and western blot. Anti-GAPDH (Abcam) (1:20,000) or anti-NS5A (Macdonald et al., 2003 (link)) (1:5000) were followed by IRDye 680RD Donkey anti-Mouse or IRDye 800CW Donkey anti-Rabbit (LI-COR BioSciences) (1:10,000) respectively. Imaging was performed using an Odyssey Imager (LI-COR).
Irdye 680rd donkey anti mouse
Manufactured by LI COR
Sourced in United States, Germany
IRDye 680RD donkey anti-mouse is a fluorescent-labeled secondary antibody used in Western blotting, immunohistochemistry, and other immunoassay techniques. It is designed to detect and visualize mouse primary antibodies.
Automatically generated - may contain errors
Lab products found in correlation
Irdye 800cw donkey anti rabbit, by LI COR (27 mentions)
Irdye 800cw goat anti rabbit, by LI COR (10 mentions)
Odyssey blocking buffer, by LI COR (6 mentions)
Irdye 800cw donkey anti mouse, by LI COR (5 mentions)
Odyssey infrared imaging system, by LI COR (5 mentions)
Odyssey imager, by LI COR (3 mentions)
Ripa buffer, by Thermo Fisher Scientific (3 mentions)
Odyssey scanner, by LI COR (3 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (3 mentions)
Irdye 680rd donkey anti rabbit, by LI COR (3 mentions)
Mouse anti β actin, by Abcam (3 mentions)
Irdye 800cw goat anti mouse, by LI COR (3 mentions)
Halt protease and phosphatase inhibitor cocktail, by Thermo Fisher Scientific (3 mentions)
Anti actin, by Merck Group (3 mentions)
Erk1 2, by Cell Signaling Technology (2 mentions)
Mouse anti β actin antibody, by Abcam (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Dc protein assay reagent, by Bio-Rad (2 mentions)
Xt sample buffer, by Bio-Rad (2 mentions)
Trans blot semi dry transfer cell, by Bio-Rad (2 mentions)
58 protocols using irdye 680rd donkey anti mouse
1
Western Blot Analysis of NS5A
2
Investigating β-catenin Regulation by Ubiquitin
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Primary antibodies used were anti-β-catenin (# 610153, BD Transduction Laboratories), anti-phospho-β-catenin pS33, pS37, pT41 (# 9561, Cell Signaling Technology), anti-non-phospho-β-catenin S33, S37, T41 (#8814, Cell Signaling Technology), anti-phospho-β-catenin pS45 (# 9564, Cell Signaling Technology), anti-StrepMAB-Classic (# 2-1507-001, IBA Life Sciences) to detect dStrepII-AXIN1, anti-AXIN1 (# 2087, Cell Signaling Technology), anti-APC (# NB100-667, Novus Biologicals), anti-GSK3β (# 12456, Cell Signaling Technology), anti-phospho-GSK3β pS9 (# 5558, Cell Signaling Technology), anti-ubiquitin K48 linkage-specific (# ab140601, Abcam), anti-ubiquitin K63 linkage-specific (# ab179434, Abcam), anti-ubiquitin P4D1 clone (# BML-PW0930, Enzo Lifesciences). Secondary antibodies for immunoblotting with detection using the Odyssey infrared imaging system (LI-COR) were IRDye 800CW donkey anti-mouse (# 926-32212, LI-COR), IRDye 800CW donkey anti-rabbit (# 926-32213, LI-COR), IRDye 680RD donkey anti-mouse (# 926-68072, LI-COR). Recombinant human poly-ubiquitin chains were obtained from commercial source: K63-linked chains (# UC-330, R&D Systems), K48-linked chains (# UC-230, R&D Systems).
3
Western Blot Analysis of Protein Samples
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Protein samples were denatured at 95 °C and resolved by 12% SDS-PAGE at 30 mA for 2 h. After transfer using Trans-Blot (Bio-Rad), nitrocellulose membranes were developed using the following primary antibodies: anti-His-tag (ab18184, dilution 1:1000), anti-DDDDK (ab49763, dilution 1:3000), anti-ALFA (FluoTag®-X2 anti-ALFA AlexaFluor 647, dilution 1:1000), anti-ZC3HAV1 (Proteintech 16820-1-AP, dilution 1:3000), anti-RPL4 (Proteintech 67028-1-Ig, dilution 1:10000), anti-RPS6 (Proteintech 14823-1-AP, dilution 1:500), anti-RYDEN (SHFL; Proteintech 27865-1-AP, dilution 1:1000). The following secondary antibodies were used: IRDye® 800CW Goat anti-rabbit (dilution 1:25000) and IRDye® 680RD Donkey anti-Mouse (dilution 1:15000; both LI-COR). Bands were visualized using an Odyssey Clx infrared imager system (LI-COR) or a Typhoon7000 (GE Healthcare).
4
HCV NS2-NS3 Protease Inhibitor Screening
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NS2-NS3-FLAG (JFH-1 isolate, genotype 2a) was purified, and proteolysis reactions were performed as previously described (Shaw et al., 2015 (link)). Compounds were added to reactions prior to addition of NS2-NS3-FLAG by dilution from DMSO stocks (final DMSO 0.75%). Reactions were incubated at room temperature for 16 h, terminated by addition of Laemmli buffer and the NS3-FLAG proteolysis product quantified by western blot using M2 anti-FLAG monoclonal antibody (Sigma Aldrich) and IRDye 680RD Donkey anti-Mouse (LI-COR Biosciences) on an Odyssey imager (LI-COR). NS3-FLAG was normalised to DMSO control and a non-linear regression fitted with Prism 6 (GraphPad) to determine 50% effective concentration (EC50).
5
Protein Extraction and Western Blot Analysis
6
Antibody Validation for Cell Signaling
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The following antibodies were used in this study: mouse anti-β-actin (Abcam, Cambridge, UK; cat# ab6276), mouse anti-γH2AX-S139 (Millipore, Burlington, MA, USA; cat# 05-636), mouse anti-c-Myc (Santa Cruz, Dallas, TX, USA; cat# sc-42), rabbit anti-cleaved PARP Asp214 (Cell Signaling, Danvers, MA, USA; cat# 9541), rabbit anti-MTH1 (Novus Biologicals, Centennial, CO, USA; cat# NB100-109), rabbit anti-p53 pS15 (Cell Signaling; cat# 9284), mouse anti-p53 (Santa Cruz; cat# sc-126), mouse anti-GAPDH (Abcam; cat# ab8245), rat anti-RPA32 (Cell Signaling; cat# 2208).
The secondary antibodies used were: goat anti-rat Alexa Fluor® 568 (Life Technologies, Carlsbad, CA, USA; cat# A-11077), goat anti-rat Alexa Fluor® 647 (Life Technologies; cat# A-21247), IRDye® 800CW donkey anti-rabbit (LI-COR, Lincoln, NE, USA; cat# 926-32213), IRDye® 680RD donkey anti-rabbit (LI-COR; cat# 926-68073), IRDye® 800CW donkey anti-mouse (LI-COR; cat# 926-32212), IRDye® 680RD donkey anti-mouse (LI-COR; cat# 926-68072).
The secondary antibodies used were: goat anti-rat Alexa Fluor® 568 (Life Technologies, Carlsbad, CA, USA; cat# A-11077), goat anti-rat Alexa Fluor® 647 (Life Technologies; cat# A-21247), IRDye® 800CW donkey anti-rabbit (LI-COR, Lincoln, NE, USA; cat# 926-32213), IRDye® 680RD donkey anti-rabbit (LI-COR; cat# 926-68073), IRDye® 800CW donkey anti-mouse (LI-COR; cat# 926-32212), IRDye® 680RD donkey anti-mouse (LI-COR; cat# 926-68072).
7
Quantifying Oxidative Stress Markers
8
Quantifying 14-3-3 Isoform Phosphorylation
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Primary antibodies included rabbit polyclonal antibody against pan 14‐3‐3 isoforms (Abcam #ab6081, Cambridge, MA), mouse monoclonal antibody against pan 14‐3‐3 isoforms (Santa Cruz #sc‐1657, Dallas, TX), mouse monoclonal antibody against 14‐3‐3θ (Abcam #ab10439), rabbit polyclonal antibody against phospho‐S58 (pS58) 14‐3‐3 (Abcam #ab51109), rabbit polyclonal antibody against phospho‐S232 (pS232) 14‐3‐3θ (Abcam #ab63369), sheep polyclonal antibody against phospho‐S185 (pS185) 14‐3‐3 (ENZO Life Science #SA‐479, Farmingdale, NY), and rabbit monoclonal antibody against phospho‐S129 αsyn (Abcam #ab168381). Horseradish peroxidase‐conjugated secondary antibodies were goat anti‐rabbit and goat anti‐sheep (Jackson Immunoresearch, West Grove, PA). Fluorescent‐conjugated secondary antibodies used were IRDye 800CW donkey anti‐rabbit and IRDye 680RD donkey anti‐mouse (LI‐COR Biotechnology, Lincoln, NE).
9
Protein Expression and TGF-β1 Secretion Analysis
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Whole cell extracts were prepared using RIPA buffer, resolved on SDS-PAGE gels, and transferred to acetate cellulose membranes. Primary antibodies used were anti-TAGLN (1:1000, Abcam plc.), anti-MYH11 (1:500, Abcam plc.), anti-ACTA2 (1:200, Sigma Corp.), anti-CNN1 (1:500, Sigma Corp.), anti-pSMAD2 (1:500, Cell Signaling Technology Inc.), anti-pS6 (1:500, Cell Signaling Technology Inc.), anti-GAPDH (1:2000, Santa Cruz Inc.). Secondary antibodies used were IRDye680RD Donkey anti-Mouse, IRDye680LT Donkey anti-Rabbit, IRDye800CW Donkey anti-Mouse, IRDye800CW Donkey anti-Rabbit (All secondary antibodies were from Licor Inc.). Licor western blot detection system was used for the dual-color imaging. ImageJ was used for the quantification of bands.
For the ELISA assay of the secreted TGF-β1, fresh medium (DMEM/F12, N2, Pen/Strep, all from Life technologies Corp.) without serum was applied to the cells and harvested after 24 h. ELISA assay was carried out using the Human TGF-beta 1 Quantikine ELISA Kit from R&D systems following the manufacture's guidelines.
For the ELISA assay of the secreted TGF-β1, fresh medium (DMEM/F12, N2, Pen/Strep, all from Life technologies Corp.) without serum was applied to the cells and harvested after 24 h. ELISA assay was carried out using the Human TGF-beta 1 Quantikine ELISA Kit from R&D systems following the manufacture's guidelines.
10
Western Blot for Phospho-Smad Proteins
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Proteins were seperated on a 7.5% Bisacrylamide gel, and transferred to a nitrocellulose membrane using wet transfer (Towbin buffer, 2.5 h 275 mA at 4 °C). After overnight incubation at 4 °C with 1:1000 polyclonal Rabbit anti P-Smad1/5 (S463/465)/Smad8 (S426/428) (Cell signaling, USA) or anti P-Smad2 (S465/467) (Cell signaling, USA), membranes were incubated with 1:1500 polyclonal Goat anti Rabbit labeled with horseradish peroxidase (HRP) (DAKO, Belgium) for 1 h. Hereafter, enhanced chemiluminescence using ECL plus (GE Healthcare, UK) was used to visualize the proteins. To visualize overexpression of constitutively active ALKs, a rabbit polyclonal antibody directed against their internal HA tag was used: HA-probe Antibody (Y-11) (Santa Cruz, USA) (1:1000). As loading control either Gapdh was stained with an anti-Gapdh mouse mAb (1G5) (Sigma Aldrich, Germany) (1:10 000) in combination with IRDye 680RD Donkey anti mouse (1:10 000) (Licor, USA) using the Odyssey detection system (Licor, USA) or Vinculin with a rabbit pAb (H300) (Santa Cruz, USA) (1:1000) in combination with HRP-labeled Goat-anti Rabbit (1:2000) (DAKO, Denmark) using ECL. Finally, blots were quantified using ImageJ.
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