The following primary antibodies were used: anti-APP CT (A8717, Sigma-Aldrich, St. Louis, MO); anti-β-amyloid, 17–24 (clone 4G8) (800701, BioLegend, San Diego, CA); anti-PS1 NT raised against the N terminus of PS1 (APS11) (ab15456, Abcam, Cambridge, MA); anti-PS1 CT raised against the C-terminus of PS1 (mAb5643, Cell Signaling Technology, Danvers, MA); anti-PS1 loop raised against the loop domain between transmembrane domains 6 and 7 of PS1 (E2000Y) (ab76083, Abcam, Cambridge, MA); anti-Syt1 (AB5600, Millipore, Temecula, CA); anti-Gapdh (mAb2118, Cell Signaling Technology, Danvers, MA); anti-MAP2 (ab5392, Abcam, Cambridge, MA); anti-His (ab18184, Abcam, Cambridge, MA); anti-V5 (ab9116, Abcam, Cambridge) and anti-FLAG M2 (F1804, Sigma-Aldrich, St. Louis, MO). Alexa Fluor 488 (ThermoScientific, Waltham, MA) and Cy3-labeled corresponding secondary antibodies (Jackson ImmunoResearch, West Grove, PA) were used for confocal microscopy imaging, and IRDye680/800- (Li-COR, Lincoln, NE) or HRP- (Jackson ImmunoResearch, West Grove, PA) conjugated ones were used for western blotting.
Irdye 680 800
Manufactured by LI COR
Sourced in United States
IRDye 680/800 are near-infrared fluorescent dyes used for labeling proteins, nucleic acids, and other biomolecules. These dyes have absorption and emission wavelengths in the 680 nm and 800 nm ranges, respectively, making them suitable for applications that require near-infrared detection.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488, by Thermo Fisher Scientific (4 mentions)
Pstat1 y701, by Cell Signaling Technology (3 mentions)
Ifit1, by Cell Signaling Technology (3 mentions)
Vinculin, by Santa Cruz Biotechnology (3 mentions)
Pstat1 s727, by Cell Signaling Technology (3 mentions)
Stat1, by Cell Signaling Technology (3 mentions)
β actin, by Merck Group (3 mentions)
Nitrocellulose membrane, by GE Healthcare (3 mentions)
Odyssey fc imaging system, by LI COR (3 mentions)
Mab5643, by Cell Signaling Technology (2 mentions)
Gapdh, by Santa Cruz Biotechnology (2 mentions)
Protease and phosphatase inhibitor, by Thermo Fisher Scientific (2 mentions)
Sars cov 2 n, by Sino Biological (2 mentions)
Ap2m1, by Abcam (2 mentions)
Pap2m1 t156, by Cell Signaling Technology (2 mentions)
Odyssey clx, by LI COR (2 mentions)
Ab5392, by Abcam (1 mentions)
Ab15456, by Abcam (1 mentions)
Ab76083, by Abcam (1 mentions)
Anti app ct, by Merck Group (1 mentions)
13 protocols using irdye 680 800
1
Amyloid Precursor Protein and Presenilins Profiling
2
Protein Immunodetection in Neural Tissues
3
Antibody Characterization for GLT-1, GLAST, and EAAT3 Detection
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The following primary antibodies were used: anti-GLT-1 raised against the peptide within 30 C-terminal amino acids from the canonical GLT-1 sequence (AB1783, EMD Millipore, Temecula, CA), and anti-GLT-1 raised against the peptide corresponding to residues 550 to the C-terminus of rat GLT-1 (ab41621, Abcam, Cambridge, MA), both recognizing GLT-1a. The GLT-1 antibody from Abcam was used in the co-IP experiments and the antibody from EMD Millipore for the immunofluorescent staining. Anti-GLAST (MABN794, Millipore, Temecula, CA); anti-EAAT3 (MAB1587, Millipore, Temecula, CA); anti-Na+/K+-ATPase (05-369, Millipore, Temecula, CA); anti-PS1 loop raised against the loop domain between transmembrane domains 6 and 7 of PS1, clone APS18 (ab15458, Abcam, Cambridge, MA) and anti-PS1 CT raised against the C-terminus of PS1 (mAb5643, Cell Signaling Technology, Danvers, MA and P7854, Sigma-Aldrich, St. Louis, MO). Alexa Fluor 488 (ThermoScientific, Waltham, MA) and Cy3-conjugated secondary antibodies (Jackson ImmunoResearch, West Grove, PA) were applied for the microscopy imaging and IRDye680/800- (Li-COR, Lincoln, NE) labeled ones were used for western blotting.
4
SARS-CoV-2 Nucleocapsid Protein Detection
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Human cell lines were grown to 70% confluence and lysed in 1X RIPA buffer (50mM Tris-HCl pH 7.4, 150mM NaCl, 1mM EDTA, 0.1% SDS, 1% NP-40, 0.25% sodium deoxycholate) supplemented with EDTA-free protease inhibitor cocktail (Roche). Proteins were separated by SDS-PAGE on 4–12% Bis-Tris gels, transferred to a nitrocellulose membranes, blocked in Intercept (PBS) blocking buffer (LI-COR) or 5% milk, and incubated with primary antibodies overnight at 4°C. Washed membranes were incubated for 45 min to 1 hour at room temperature in secondary antibody solution (LI-COR IRDye 680, 800; 1:10,000 in 5% milk or Intercept (PBS) blocking buffer), imaged on an Odyssey® CLx scanner, and analyzed using the Image Studio Software. Antibodies were used at the following dilutions: ACE2 (R&D Systems #AF933, 1:200), β-actin (Sigma #A5316, 1:5000 or Santa Cruz Biotech #sc8432, 1:500), Vinculin (Santa Cruz #sc-73614, 1:2000), pSTAT1-Y701 (Cell Signaling #9167, 1:1000), pSTAT1-S727 (Cell Signaling #8826, 1:1000), STAT1 (Cell Signaling #14994, 1:1000), MX1 (Cell Signaling #37849, 1:1000), IFIT1 (Cell Signaling #14769, 1:1000), STING (Cell Signaling #13647S, 1:1000), cGAS (Cell Signaling #15102S, 1:1000), TBK1 (Cell Signaling #38066S, 1:1000), IRF3 (Cell Signaling #4302S, 1:1000), SARS-CoV-2 Nucleocapsid (Sino Biological # 40588-T62, 1;1000 or # 40143-MM05, 1:1000).
5
Western Blot Analysis of CA IX Protein
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Overall protein and phosphorylation were analyzed by Western blotting using mouse CA IX antibody (1:2,000; BioScience, Bratislava, Slovakia) and mouse β-actin, ERK1/2, p38, or phospho-specific antibodies (1:1,000; Cell Signaling Technology, Danvers, MA, USA). In brief, cells were washed in PBS and lysed with Laemmli buffer [60 mM Tris-HCl (pH 6.8), 2% SDS, 10% glycerol, 5% β-mercaptoethanol, 0.005% bromophenol blue], separated by SDS-PAGE, and transferred to nitrocellulose membranes. Membranes were probed for rabbit phospho-ERK1/2 (Thr202/Tyr204) and overall mouse ERK1/2 or mouse phospho-p38 (Thr180/Tyr182) and rabbit p38 antibody simultaneously using LI-COR multiplex detection with secondary antibodies labeled with distinct near-infrared fluorescent dyes (1:40,000; IRDye 680/800; LI-COR Bioscience, Lincoln, NE, USA). Quantitative analysis was performed with the Image Studio Lite software (LI-COR Bioscience). Expression of CA IX was normalized to β-actin.
6
Immunoblot Protein Detection and Analysis
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Cell extract samples were separated on 12% SDS-PAGE gels before transfer to nitrocellulose membranes (GE Healthcare). Membranes were blocked in PBS containing 5% milk before probing with primary antibodies at concentrations listed in S2 Table . Following overnight incubation at 4°C, membranes were washed and treated with IRDye-680/800- (LI-COR) or horseradish peroxidase-conjugated (GE Healthcare) secondary antibodies. Infrared and chemiluminescent bands were visualized using a LI-COR Odyssey Fc Imaging System. Band intensities were measured using LI-COR Image Studio software. Densitometries of proteins-of-interest were normalized to GAPDH, tubulin, and/or actin loading controls.
7
Western Blot Analysis of ERK Signaling
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Whole cell or tissue lysates were prepared in RIPA buffer supplemented with protease and phosphatase inhibitors, and samples sonicated to achieve optimum lysis. Protein concentrations were quantitated using the BCA assay, and 20 μg of lysates were loaded into 8% SDS-polyacrylamide gels. Gels were resolved at 80V for 120 mins, transferred onto nitrocellulose membrane for 70 mins at 0.35A with transfer buffer containing 20% methanol, and blocked for one hour at room temperature with 5% w/v BSA in 0.1% TBST. Membranes were incubated with primary antibodies in 5% w/v BSA in 0.1% TBST. Antibodies used in this study are: Total ERK (1:1000, Cell Signaling #9102), phospho ERK (1:1000, Cell Signaling #4695), GAPDH (1:5000, Santa Cruz sc-69778), anti-mouse and anti-goat secondary antibodies (1:10000, LiCOR IRDye 680/800 Cat. 925-68070/926-32211). Blots were imaged using the LiCOR Odyssey Fc system.
8
Detailed Antibody Protocol for Desmosomes
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Antibodies used were: H145 Dsg2 (1:10,000), Cav1 (1:40,000), and GAPDH (1:3,000; Santa Cruz, Santa Cruz, CA); Actin (1:100,000; Calbiochem, Billerica, MA);; Flotillin 1, c-Src, P-c-Src Tyr416, P-c-Src Tyr527, EGFR, P-EGFR Tyr1173, P-EGFR Tyr1045, P-EGFR Tyr845, P-Erk1/2 (Thr202/Tyr204), P-AKT (Thr308), P-Stat3 (Tyr705) (1:1,000; Cell Signaling, Danvers, MA); 10D2 Dsg2 (1:2), 27B2 Dsg1 (1:100), 5H10 Dsg3 (1:100), 18D4 Dsg4 (1:100), desmocollin-2/3 7G6 (1:10,000), 20F6 desmoplakin (1:50), 14B11 plakophillin-1 (1:50), 8H6 plakophillin-2 (1:50), 19A5 plakophillin-3 (1:100), 11E4 plakoglobin (1:100), 4A2 E-cadherin (1:2,500), 6F9 β-catenin (1:1,000) [72 (link)]; Secondary antibodies: Alexa Fluor-488 & −594 (1:400, Molecular Probes, Eugene, OR); HRP (1:5,000; Jackson Labs, Bar Harbor, ME); IRDye 680 & 800(1:20,000; LI-COR Biosciences, Lincoln, NE); Secondary antibodies for STED-imaging: Mega520-conjugated anti-Rabbit (Sigma-Aldrich) and Star635P-conjugated anti-Mouse (Abberior GmbH, Göttingen, Germany).
9
Western Blot Analysis of SARS-CoV-2 Infection
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Human cell lines were grown to 70% confluence and lysed in RIPA (10% glycerol, 50mM Tris-HCl pH 7.4, 150mM NaCl, 2mM EDTA, 0.1% SDS, 1% NP40, 0.2% sodium deoxycholate) containing protease and phosphatase inhibitors (Thermo Scientific). Proteins were separated by SDS-PAGE, transferred to a nitrocellulose membrane, blocked in 5% milk, and incubated with primary antibodies overnight at 4°C. Washed membranes were incubated for 45 min at room temperature in secondary antibody solution (LI-COR IRDye 680, 800; 1:10,000 in 5% milk), imaged on an Odyssey® CLx, and analyzed using Image Studio Software. Antibodies were used at the following dilutions: ACE2 (R&D Systems #AF933, 1:200), β-actin (Sigma #A5316, 1:5000), Vinculin (Santa Cruz #sc-73614, 1:2000), AAK1 (Bethyl #A302–146A, 1:1000), AP2M1 (Abcam #ab75995, 1:1000), pAP2M1-T156 (Cell Signaling #3843, 1:1000), SARS-CoV-2-N (Sino Biological #40588-T62, 1:500), pSTAT1-Y701 (Cell Signaling #9167, 1:1000), pSTAT1-S727 (Cell Signaling #8826, 1:1000), STAT1 (Cell Signaling #14994, 1:1000), MX1 (Cell Signaling #37849, 1:1000), IFIT1 (Cell Signaling #14769, 1:1000), pIKKα/β-S176/180 (Cell Signaling #2697, 1:1000), IKKα (Cell Signaling #11930, 1:1000), IKKβ (Cell Signaling #8943, 1:1000), pNFKB p65-S536 (Cell Signaling #3033, 1:1000), NFKB p65 (Cell Signaling #8242, 1:1000),
10
Protein Extraction and Western Blot Analysis
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For preparation of cell extracts, MTFs cultured in 6-well plates were collected in phosphate-buffered saline (PBS) containing 1mM EDTA, centrifuged at 750 x g for 5.5min at 4°C, and lysed in 25mM HEPES (pH 7.4), 100mM NaCl, 1% Triton-X-100, 1mM EDTA, 1mM Na3VO4, 1mM NaF, 1mM PMSF, and 10μg/ml each of aprotinin, leupeptin, pepstatin, and chymostatin on ice. Lysates were mixed with Laemmli sample buffer, boiled, and centrifuged. Extract samples were separated on 12% SDS-PAGE gels before transfer to nitrocellulose membranes (GE Healthcare). Membranes were blocked in PBS containing 5% milk before probing with primary antibodies at concentrations listed in Table S2 . Following overnight incubation at 4°C, membranes were washed and treated with IRDye-680/800- (LI-COR) or horseradish peroxidase-conjugated (GE Healthcare) secondary antibodies. Infrared and chemiluminescent bands were visualized using a LI-COR Odyssey Fc Imaging System. Band intensities were measured using LI-COR Image Studio software. Densitometries of proteins-of-interest were normalized to GAPDH, tubulin, and/or actin loading controls.
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