293 T cells were transfected with FOLR1-myc constructs using Lipofectamine 3000, incubated for 48 hr and lysed in Pierce RIPA buffer (Thermo #89901) containing Protease Inhibitor cocktail (Roche #11697498001). Lysate protein concentration was determined by BCA assay and electrophoresis was performed on a 10% Tris-glycine gel. Protein was transferred to a PVDF membrane, blocked in Odyssey blocking buffer and incubated o/n at 4°C with 1:1000 Rabbit anti-myc (Abcam ab9106) and 1:1000 Mouse anti-Tubulin (Sigma #T6199) antibodies). Membranes were washed and incubated for 1 hr in 1:15000 goat anti-rabbit IRDye 800CW (LICOR # 926–32211)and 1:15000 goat anti-mouse IRDye 680Rd (LICOR, #926–68070) and visualized on LICOR Odyssey imaging system.
Irdye 680rd goat anti mouse
Manufactured by LI COR
Sourced in United States, Germany
IRDye 680RD goat anti-mouse is a fluorescently labeled secondary antibody used for detection in Western blotting, immunohistochemistry, and other applications. It is specific for mouse primary antibodies and emits fluorescence in the near-infrared range.
Automatically generated - may contain errors
Lab products found in correlation
Irdye 800cw goat anti rabbit, by LI COR (88 mentions)
Irdye 680rd goat anti rabbit, by LI COR (16 mentions)
Odyssey clx imaging system, by LI COR (14 mentions)
Irdye 800cw goat anti mouse, by LI COR (11 mentions)
Odyssey imaging system, by LI COR (9 mentions)
Odyssey infrared imaging system, by LI COR (9 mentions)
Nupage bis tris gel, by Thermo Fisher Scientific (9 mentions)
Odyssey clx, by LI COR (8 mentions)
Odyssey clx system, by LI COR (8 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (7 mentions)
Nitrocellulose membrane, by Thermo Fisher Scientific (7 mentions)
Odyssey blocking buffer, by LI COR (6 mentions)
Image studio software, by LI COR (6 mentions)
Anti β actin, by Merck Group (6 mentions)
Irdye 800cw donkey anti rabbit, by LI COR (6 mentions)
Protease inhibitor cocktail, by Roche (5 mentions)
Immobilon fl pvdf membrane, by Merck Group (5 mentions)
Nitrocellulose membrane, by Bio-Rad (5 mentions)
Image studio, by LI COR (5 mentions)
Cell lysis buffer, by Cell Signaling Technology (5 mentions)
148 protocols using irdye 680rd goat anti mouse
1
FOLR1 Protein Expression Analysis
2
Protein Abundance Detection Protocol
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Protein extractions were performed as previously described44 (link),49 . Primary antibodies HAS2 (Santa Cruz Biotechnology, sc-34068) (1:200 dilution), HSL (Santa Cruz Biotechnology, sc-74489) (1:200 dilution), α Tubulin (Santa Cruz Biotechnology, sc-53030) (1:200 dilution), Actin (Sigma #A4700) (1:1000 dilution), Adiponectin (homemade) (1:1000 dilution) were used. Protein abundance was detected using the one of the following secondary antibodies: Goat anti-Mouse IRDye 680RD (Li-cor 926-68070), Goat anti-rabbit IRDye 800CW (Li-cor 925-32211) or Goat anti-Rat DyLight 800 (Thermo Fisher SA5-10024) at 1:10,000 dilutions. Antibody decorated membranes were then visualized on a Li-Cor Odyssey infrared scanner (Li-Cor Bioscience). The scanned data were analyzed using Odyssey Version 3.0 software (Li-Cor Bioscience).
3
Western Blot Analysis of Tissue Proteins
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Brain, eyes or retinas from ≥three animals were homogenized in cold RIPA buffer containing proteases inhibitors (Roche Complete Mini-EDTA free protease inhibitors, 100 μM leupeptin, 100 mM NaVO4, 20 mM NaF) using a BeadBug™ Microtube Homogenizer (Benchmark Scientific Model D1030E) as in10 (link). 50 μg extract samples were electrophoresed on 4–12% SDS- polyacrylamide gels and transferred to nitrocellulose membranes (Li-COR #926–31090). Membranes were incubated in Odyssey® Blocking Buffer (Li-COR, #P/N 927–50000) for 1 h at room temperature, treated with primary antibody (Supp. Table S2 ) in Blocking Buffer +0.1% Tween 20 for 12–6 h at 4 °C and incubated for 1 h at room temperature in secondary antibody (goat anti-rabbit IRDye 800CW or goat anti-mouse IRDye680RD; LiCOR). Membranes were scanned on Odyssey Fc Dual-Mode Imaging System (Li-COR) and data quantified using Image Studio Software (Li-COR), according to manufacturer’s protocols. Actin was used as a reference protein for quantitative immunoblots. All commercially available antibodies used for immunoblots and immunofluorescence experiments are listed in Supp. Table S2 . Affinity purified rabbit polyclonal anti-Ndr2 antibody was generated using an Ndr2-specific peptide antigen (QPVPNTTEPDYKSK, corresponding to amino acids 421–434) (YenZym Antibodies LLC, San Francisco, CA), as previously described29 (link).
4
Western Blot Analysis of Transcription Factors
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Protein was extracted from 5 ml of log-phase culture using a TCA/NaOH precipitation as described in Volland et al. [57 (link)]. Equal concentrations of protein (25 μg) were run on a NuPAGE 4–12% Bis-Tris gel (Invitrogen, #NP0323BOX) and transferred to a nitrocellulose membrane (Bio-rad 0.2 µm, #LC2009). Primary antibodies used for western blotting: rat anti‐FLAG (Agilent, #200474), mouse anti‐PGK1 (Abcam, #Ab113687), mouse anti‐Rpb3 (Biolegend, #665004), rat anti-S2P (Chromotek, #3E10), rat anti-S5P (Chromotek, #3E8), Rabbit anti-Spt5-P (#1761, kind gift from Prof. Steven Hahn) [27 (link)] and Rabbit anti-Spt5 (kind gift from Prof. Grant Hartzog) [58 (link)]. Secondary antibodies used for western blotting: goat anti-mouse IRDye680RD (LI-COR, #926-68070), goat anti-rat IRDye800RD (LI-COR, #926-32219), goat anti-rabbit IRDye800RD (LI-COR, #925-32211). The Odyssey infrared imaging system (LI-COR Bioscience) was used to visualize proteins-of-interest, and signals quantified by the median method of the Odyssey software. For quantitation, data were normalized against the PGK1 signal unless otherwise stated in the figure legend.
5
Quantitative Western Blot Analysis
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Protein was extracted using the RNA/DNA/Protetin Purification Kit (Norgen). Protein concentration was measured using Coomassie Plus (Pierce). Equal amounts of total protein were mixed with sample buffer (Invitrogen) and reducing agent (Invitrogen). Samples were heated at 70°C for 10 minutes and loaded onto a 4–12% Bis-Tris NuPAGE gel (Invitrogen). Gels were run on a NuPAGE electophoresis system (Invitrogen) at 200 V for 50 minutes in MOPS running buffer. Samples were then transferred onto an Immobilon-FL PVDF membrane (Millipore) at 30 V for 1 hour and blocked with Odyssey blocking buffer (LiCore). The following antibodies were used: rabbit-anti-REDD1 (Proteintech), mouse-anti-actin (Sigma), goat-anti-rabbit-IRDye 800CW (LiCor) and goat-anti-mouse-IRDye 680RD (LiCor). The membranes were imaged on the Odyssey CLx (LiCor) and analyzed using the Image Studio software (LiCor).
6
Quantifying Mitochondrial TFAM Levels
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Western blot was used to assess intramitochondrial and extramitochondrial (= cytonucleoplasmic) TFAM protein levels. Equal amounts of protein were separated on a 4–20% polyacrylamide gradient gel (Bio-Rad, Hercules, CA) and subsequently blotted onto a nitrocellulose membranes (Bio-Rad). The membranes were probed with the primary antibodies against TFAM (1:200; sc-376672, Santa Cruz Biotechnology, Dallas, TX), TNF receptor-associated protein 1 (1:500; Sigma-Aldrich), and beta actin (1:10,000¸ Millipore, Temecula, CA) for five hours at room temperature. After incubation of secondary antibodies (1:10,000; Goat-anti-mouse, IRDye680RD, and 1:10,000; donkey-anti-rabbit, IRDye 800cw, both Li-cor Biosciences, Lincoln, NE) for one hour at room temperature, protein bands were visualized with an Odyssey Scanner (Li-cor Biosciences) and the densitometry was determined using Image J 2.0 software (National Institutes of Health, Bethesda, MD). TRAP1 was used for normalization since its concentration proved to be stable in mitochondria during LPS stimulation (Supplementary Figure 3 ).
7
Spinal Protein Expression Analysis
8
Western Blot Analysis of EMT Markers
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Western blot analysis (WB) was carried out essentially as previously described in (63 (link)). The detection and quantification were performed with Odyssey Clx System (LI-COR Biosciences) through the Image Studio Software, or by traditional ECL detection. The following antibodies were used: anti-ZEB1-1642 (from Dr. Douglas Darling Lab), anti-ZEB1 (Santa Cruz,# sc-25388), anti-E-cadherin (BD Biosciences,# 610182), anti-vimentin (Cell Signaling, #5741), anti-SNAIL (Cell Signaling, #3879), anti β-catenin (Cell Signaling, #8480), anti ZO-1 (Cell Signaling, #8193), anti-cytokeratin 18 (Cell Signaling, #4548), anti-phospho serine/threonine (Abcam, #ab9337), anti-phospho-substrates antibodies kit Cell Signaling, #9920) anti-GFP (Abcam, #ab290), anti-PKCα (Santa Cruz, # sc-208), anti-PKCδ (Cell Signaling, #2058) anti-PKCε (Santa Cruz,# sc-1681), anti- Phospho-PKCα/β II (Cell Signaling, #9375) anti-α-tubulin (Sigma-Aldrich, #T5168); anti-β-actin (Sigma-Aldrich, #A2228); anti-PCNA (Santa Cruz, #sc56). As secondary antibodies we used anti-rabbit-Alexa-Fluor 594 (Molecular Probes), anti-rabbit HRP (Cell Signaling, #7074), anti-mouse HRP (Cell Signaling, #7076), goat anti-mouse IRDye 680RD and goat anti-rabbit IRDye 800CW (LI-COR Biosciences).
9
Cell Surface Biotinylation of Mouse Brain Slices
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Cell surface biotinylation of mouse brain slices was conducted as published [62 (link)]. After euthanization, mouse brains were freshly isolated and sliced into 1 mm coronal sections. Brain slices were incubated in 1 mg/ml sulfo-NHS-SS-biotin (ThermoFisher) in pre-chilled oxygenated aCSF on ice for 30 min and then washed twice with 50 mM glycine and three times with 1 mg/ml BSA in pre-chilled oxygenated aCSF. Hippocampus was dissected from slices, extracted and the biotinylated proteins purified using NeutrAvidin beads (ThermoFisher) and processed for Western blot with following antibodies: rabbit anti CLC-3 (Rabbit, 1:1000, Alomone, ACL-001), Rabbit anti-Na-K ATPase (1:10,000, Abcam, ab76020). Blots were developed with goat anti-mouse IRDye 680RD and goat anti-rabbit IRDye 800CW (1:5000, LI-COR) and Odyssey CLx imager (LI-COR) and quantitated using Image Studio (LI-COR).
10
FGFR4 and CAR Moiety Detection
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Expression of FGFR4-His, anti-FGFR4 Nb-Fc and CAR moiety were detected by western blot assay after transfection of encoding vector to HEK293T cells. After 2 days, the supernatant was collected and the transfected cells were lysed using NP40 buffer containing protease inhibitors (Sigma). The supernatant or lysate was boiled after adding to SDS loading buffer. The proteins were separated using 4–12% SurePAGE (Genscript) and transferred to the nitrocellulose transfer membrane for immunoblotting. Expression of FGFR4-His was detected by anti-His tag antibody (Proteintech, 66005-1-Ig). Expression of CAR moiety was detected by anti-Flag tag antibody (MBL, M185-3). Other antibodies we used included anti-GAPDH antibody (Cat No. 10494-1-AP, Proteintech), goat anti-rabbit IRDye 800CW (Cat No. 926-32211, Li-cor), and goat anti-mouse IRDye 680RD (Cat No. 926-68070, Li-cor). Images were acquired by using the two-color infrared laser imaging system Odyssey (Li-cor) and analyzed through using Image Studio Lite Ver 4.0 software (Li-cor).
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