T-cells from co-culture experiments (1 × 105 T-cells seeded) were harvested with one part of 1× PBS and one part of Novex® buffer (Thermo Fisher Scientific) containing 3% dl -dithiothreitol (Sigma-Aldrich). Samples were run on 10% SDS PAGE gels and then blotted on an Amersham protan nitrocellulose transfer membrane (Sigma-Aldrich). Phospho-protein phosphatase 2A (pPP2A) (1:7,000, clone E155; Abcam, Cambridge, UK), protein phosphatase 2A (PP2A) (1:1,000), pAkt (Ser 473, 1:2,000, clone D9E), pAkt (Thr 308, 1:1,000, clone C31E5E), Akt (1:1,000, clone C67E7, all Cell signaling technology, Danvers, MA, USA), and vinculin (clone hVIN-1, 1:40,000 Sigma-Aldrich) protein expressions were determined by immunoblotting of CD3+ T-cells at intervals up to 96 h after allogeneic stimulation with DCs. Western blots for pPP2A and pAKt (Ser473 and Thr308) were developed by chemiluminescence imaging using the Super signal west femto maximum sensitivity substrate (Thermo Fisher Scientific) and chemiluminescent detection films (Sigma-Aldrich). Western blots for PP2A, Akt, and vinculin were developed by fluorescence imaging using Goat-anti-mouse IgG Dylight 800 and Goat-anti-rabbit IgG Dylight 800 (both Thermo Scientific) on an Odyssey Fluorescence imager (Licor, Licoln, NE, USA). Western blots were analyzed by densitometry using the ImageJ software.
Goat anti rabbit igg dylight 800
Manufactured by Thermo Fisher Scientific
Sourced in Australia
Goat anti-rabbit IgG DyLight 800 is a secondary antibody used to detect and quantify rabbit primary antibodies. It is conjugated with the DyLight 800 fluorescent dye, which emits light in the near-infrared range, making it suitable for immunofluorescence and Western blot applications.
Automatically generated - may contain errors
Lab products found in correlation
Odyssey infrared imaging system, by LI COR (6 mentions)
Goat anti mouse igg dylight 680, by Thermo Fisher Scientific (5 mentions)
Odyssey clx system, by LI COR (2 mentions)
Image studio lite v5, by LI COR (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Complete protease inhibitor cocktail, by Merck Group (2 mentions)
Iblot2 transfer stacks pvdf membrane, by Thermo Fisher Scientific (2 mentions)
Anti gfp, by Thermo Fisher Scientific (2 mentions)
Anti glucose 6 phosphate dehydrogenase g 6 pdh, by Merck Group (2 mentions)
Rabbit anti actin, by Merck Group (2 mentions)
Rabbit anti sting, by Cell Signaling Technology (2 mentions)
Odessy scanner, by LI COR (2 mentions)
Rabbit anti tubulin, by Cell Signaling Technology (2 mentions)
Mouse anti actin, by Thermo Fisher Scientific (2 mentions)
Mouse anti tmprss2, by Santa Cruz Biotechnology (2 mentions)
Rabbit anti irf1, by Cell Signaling Technology (2 mentions)
Mouse anti cathepsin l, by Santa Cruz Biotechnology (2 mentions)
Rabbit anti mavs, by Thermo Fisher Scientific (2 mentions)
Mouse anti rig 1, by Adipogen (2 mentions)
Rabbit anti mda 5, by Proteintech (2 mentions)
13 protocols using goat anti rabbit igg dylight 800
1
Phospho-Proteomics of Activated T-cells
2
Western Blot Analysis of STAT3 Phosphorylation
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Cells were rinsed twice, with cold PBS, and lysed in ice-cold RIPA buffer (150 mM sodium chloride, 1.0% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris, pH 8.0; Thermo Fisher Scientific), supplemented with complete Protease Inhibitor Cocktail (Sigma Aldrich) and PhosStop (Thermo Fisher Scientific). Protein concentrations were determined using Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) as per the manufacturer’s protocol. Up to 30 µg of protein per sample was loaded on NuPAGE 8–12% Bis-Tris protein gels (Thermo Fisher Scientific) and transferred onto iBlot2 Transfer Stacks PVDF membrane (Thermo Fisher Scientific). Membranes were blocked with Odyssey Blocking buffer (Milennium Science, Victoria, Australia) for 1 h, probed with rabbit anti-STAT3p antibody (1:200; 9145, Cell Signaling Technology) and mouse anti-β-actin (1:5000; NB600-501, Novus Biologicals) overnight, followed by detection with goat anti-rabbit IgG DyLight 800 (1:20,000; Thermo Fisher Scientific) and goat anti-mouse IgG DyLight 600 (1:20,000; Thermo Fisher Scientific). Bands were visualized using the Odyssey CLx system (LI-COR Biosciences, Nebraska, USA) and analyzed with Image Studio Lite V5.2 (LI-COR Biosciences).
3
Western Blot Analysis of Protein Localization
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The indicated strains were grown to mid–log phase in yeast extract, peptone + 2% (wt/vol) dextrose (YPD) media. For Supplemental Figure S2, B and C, 0.5 nM estradiol and 0.05 nM estradiol were added, as indicated, to drive expression of GalS::MMR1-yEGFP and GalS::MMR1ΔCC-yEGFP, respectively. Cells (1.0 OD) were harvested, and whole cell extracts were prepared using a NaOH lysis and trichloroacetic acid (TCA) precipitation procedure. Each TCA pellet was resuspended in 50 µl MURB (100 mM MES, pH 7, 1% SDS, and 3 M urea). Whole cell extracts were analyzed by SDS–PAGE followed by Western analysis using anti-GFP (Invitrogen), anti-glucose-6-phosphate dehydrogenase (G-6-PDH; Sigma-Aldrich), or anti-phosphoglycerate kinase (PGK; Life Technologies) as the primary antibodies and goat anti-rabbit IgG DyLight 800 or goat anti-mouse IgG DyLight 680 (Thermo Fisher Scientific) as the secondary antibodies. The immunoreactive bands were detected with the Odyssey Infrared Imaging System (LI-COR Biosciences).
4
Western Blot Protein Analysis Protocol
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The separation of proteins was performed, accordingly to their size, in denaturing conditions, through SDS-PAGE vertical electrophoresis. Proteins were transferred to PVDF Immobilon-P or Immobilon-FL membranes (Millipore) as previously reported [28 (link), 36 , 47 (link), 67 (link)]. PDVF membranes were incubated with the specific primary antibody (Table 1 ). The secondary antibodies Goat Anti-Mouse IgG, DyLight 680 (red colored) and Goat Anti-Rabbit IgG, DyLight 800 (green colored) (Thermo Scientific) were incubated at 1:10,000 dilutions in TBS-T, during 1 h (in the dark). Next, membranes were washed with TBS-T (3×, 10 min washes) and scanned in the LI-COR Odyssey Infrared Imaging System (LI-COR Biosciences; Lincoln, NE, USA) that detects the fluorescence associated with the secondary antibody. The secondary antibodies were either Anti-Mouse IgG-Horseradish Peroxidase-Linked Species-Specific Whole Antibody (Amersham Biosciences; Amersham, UK) or Anti-Rabbit IgG-Peroxidase Conjugate (Sigma-Aldrich). In these cases, after a 1 h incubation with the secondary antibody (1:10,000), the luminescence was detected with the ECL Western Blotting Detection Reagent.
5
Auxin-Induced Protein Depletion Assay
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For Fig. S3 A, mitoAID and mitoAID Δkar9 cells expressing Tir1 and W303 Δkar9 cells were grown as described in the Imaging section. Equivalent ODs of cells were taken from all samples before and 3 h after the addition of DMSO or 1 mM auxin. For Fig. S3 D, mitoAID NUM1-yEGFP cells expressing Tir1 were grown as described in the Imaging section. Equivalent ODs of cells were taken before and 30 min after the addition of DMSO or 1 mM auxin. Whole-cell extracts were prepared using a NaOH lysis and TCA precipitation procedure. Each TCA pellet was resuspended in 50 µl MURB (100 mM MES, pH 7, 1% SDS, and 3 M urea). Whole-cell extracts were analyzed by SDS-PAGE followed by Western analysis using anti-FLAG antibody (Sigma-Aldrich) and anti–glucose-6-phosphate dehydrogenase (G-6-PDH) antibody (Sigma-Aldrich) as the primary antibodies and goat anti–mouse IgG DyLight 680 and goat anti–rabbit IgG DyLight 800 (Thermo Fisher Scientific), respectively, as the secondary antibodies. The immunoreactive bands were detected with the Odyssey Infrared Imaging System (LI-COR Biosciences).
6
Visualizing Cx30.3 Expression and UPR Activation
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REKs engineered to transiently express FLAG-tagged Cx30.3 or Cx30.3 mutants were subjected to SDS-PAGE and Western blotting as previously described (Au et al., 2020 (link); Lucaciu et al., 2022 (link)). Blots were labeled with rabbit anti-FLAG (1:3000) and mouse anti-GAPDH (1:5000; MilliporeSigma, Cat# MAB374) antibodies overnight at 4°C. Primary antibodies were removed, blots were washed, and then labeled with goat anti-mouse IgG AlexaFluor 680 (1:10000) (ThermoFisher, Cat# A-21057) and goat anti-rabbit IgG DyLight™ 800 (1:10000, ThermoFisher, Cat# SA5-10036) for 45 min at room temperature. Secondary antibodies were removed, blots washed, and protein bands were subsequently visualized using the LI-COR Odyssey infrared imaging system. In some cases, control and cells treated with 2 μg/mL of tunicamycin (MilliporeSigma, Cat# 11089-65-9) for 24 h or cells engineered to express Cx30.3 or mutants were immunoblotted for BiP/GRP78 (1:2000) to probe for UPR activation.
7
Protein Expression Profiling Using Western Blot
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To assess expression of endogenous or transduced proteins, cell lysates containing 30–40 μg total protein were separated by SDS-PAGE, transferred to nitrocellulose membranes and the membranes were probed with the following antibodies: mouse anti-TMPRSS2 (Santa Cruz, #515727, 1:1000), mouse anti-Cathepsin-L (Santa Cruz, #32320, 1:1000), goat anti-ACE-2 (R&D systems, #AF933, 1;1000), rabbit anti-STING (Cell Signaling, #13647, 1:1000), rabbit anti-MAVS (Thermo Fisher, #PA5–17256, 1:1000), mouse anti-RIG-I (AdipoGen, #20B-0009, 1:1000), rabbit anti-MDA-5 (Proteintech, #21775–1-AP, 1:1000), rabbit anti-UNC93B1 (Invitrogen, #PA5–83437, 1:1000), rabbit anti-IRF1 (Cell Signaling, #8478S, 1:1000). Specific staining was visualized with secondary antibodies, goat anti-mouse-IgG-DyLight 680 (Thermo Scientific, #35518, 1:20000), goat anti-rabbit-IgG-DyLight 800 (Thermo Scientific, #SA5–35571, 1:20000), or a donkey anti-goat-IgG-IR-Dye 800 (Licor, #926–32214, 1:20000). As loading controls, actin or tubulin expression was probed using a rabbit anti-actin (Sigma-Aldrich, A2066, 1:5000), mouse anti-actin (Invitrogen, #AM4302, 1:5000), or rabbit anti-tubulin (Cell Signaling, #3873, 1;5000). Membranes were scanned with an Odessy scanner (Li-Cor).
8
Western Blot Analysis of GFP-Tagged Proteins
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The indicated strains were grown to midlog phase in yeast extract, peptone + 2% (wt/vol) dextrose (YPD) media. 1.0 OD of cells were harvested, and whole cell extracts were prepared using a NaOH lysis and TCA precipitation procedure. Each TCA pellet was resuspended in 75 µl MURB (100 mM MES, pH 7, 1% SDS, and 3 M urea). Whole cell extracts were run on 3–8% Tris-Acetate gels (ThermoFisher, https://www.thermofisher.com/order/catalog/product/EA03785BOX ) in denaturing conditions followed by western analysis using anti-GFP (ThermoFischer, https://www.thermofisher.com/antibody/product/GFP-Tag-Antibody-Polyclonal/A-11122 ) and anti-glucose-6-phosphate dehydrogenase (G-6-PDH; Sigma-Aldrich, https://www.sigmaaldrich.com/catalog/product/sigma/a9521?lang=en®ion=US ) as the primary antibodies and goat anti-rabbit IgG DyLight 800 (ThermoFisher, https://www.thermofisher.com/antibody/product/Goat-anti-Rabbit-IgG-H-L-Secondary-Antibody-Polyclonal/SA5-35571 ), respectively, as the secondary antibodies. The immunoreactive bands were detected with the Odyssey Infrared Imaging System (LI-COR Biosciences).
9
Analyzing Autophagy in BCG-Infected THP-1 Cells
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THP-1 cells were infected with BCG with/without 100 nM 7α,25-OHC and with/without 10 µM GSK682753 and lysed at 6 or 24 h post infection (p.i.) in ice-cold RIPA buffer (150 mM sodium chloride, 1.0% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris, pH 8.0; Thermo Fisher Scientific), supplemented with complete Protease Inhibitor Cocktail (Sigma Aldrich) (120 µl RIPA/1 × 106 Cells). Protein concentrations were determined using Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) as per manufacturer’s protocol. 10 µg of protein per sample was loaded on NovexTM 10–20% Tris-Glycine protein gels (Thermo Fisher Scientific) and transferred onto iBlot2 Transfer Stacks PVDF membrane (Thermo Fisher Scientific). Membranes were blocked with Odyssey Blocking buffer (Milennium Science, Victoria, Australia) for 2 h, probed with rabbit anti-human LC3B (1:1,000, Sigma L7543) and rabbit anti-human GAPDH (1:2,500, Abcam 9485) overnight, followed by detection with goat anti-rabbit IgG DyLight 800 (1:20,000; Thermo Fisher Scientific). Bands were visualized using the Odyssey CLx system (LI-COR Biosciences, Nebraska, USA) and analyzed with Image Studio Lite V5.2 (LI-COR Biosciences).
10
Western Blot Analysis of Antiviral Proteins
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To assess expression of endogenous or transduced proteins, cell lysates containing 30–40 μg total protein were separated by SDS-PAGE, transferred to nitrocellulose membranes and the membranes were probed with the following antibodies: mouse anti-TMPRSS2 (Santa Cruz, #515727, 1:1000), mouse anti-Cathepsin-L (Santa Cruz, #32320, 1:1000), goat anti-ACE2 (R&D systems, #AF933, 1;1000), rabbit anti-STING (Cell Signaling, #13647, 1:1000), rabbit anti-MAVS (Thermo Fisher, #PA5-17256, 1:1000), mouse anti-RIG-I (AdipoGen, #20B-0009, 1:1000), rabbit anti-MDA-5 (Proteintech, #21775-1-AP, 1:1000), rabbit anti-UNC93B1 (Invitrogen, #PA5-83437, 1:1000), rabbit anti-IRF1 (Cell Signaling, #8478S, 1:1000). Specific staining was visualized with secondary antibodies, goat anti-mouse-IgG-DyLight 680 (Thermo Scientific, #35518, 1:20000), goat anti-rabbit-IgG-DyLight 800 (Thermo Scientific, #SA5-35571, 1:20000), or a donkey anti-goat-IgG-IR-Dye 800 (Licor, #926–32214, 1:20000). As loading controls, actin or tubulin expression was probed using a rabbit anti-actin (Sigma-Aldrich, A2066, 1:5000), mouse anti-actin (Invitrogen, #AM4302, 1:5000), or rabbit anti-tubulin (Cell Signaling, #3873, 1:5000). Membranes were scanned with an Odessy scanner (Li-Cor).
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