SEP-B2AR, SEP-MOR, SEP-GluR1, and TfR-SEP have been previously described (Yudowski et al., 2006 (link), 2007 (link), 2009 (link); Yu et al., 2010 (link)). To produce mCherry-TfR-SEP, mCherry was amplified with PCR using primers that include Agel flanking sites: 5'-GCGCGCGCGCACCGGTAGTGAGCAAGGGCGAGGAGGATAACA-3' and 5'-GCGCGCGCGCACCGGTATCTTGTACAGCTCGTCCATGCCG-3'. The fragment was then cloned into TfR-SEP. Clathrin light chain DsRed (CLC-DsRed) was a gift from Wolf Almers (Vollum Institute, Oregon). All chemicals were purchased from Sigma-Aldrich except dyngo-4a, which was purchased from Abcam (Cambridge, MA).
Dyngo 4a
Manufactured by Abcam
Sourced in United Kingdom, Germany, Canada
Dyngo-4a is a chemical compound that functions as a dynamin inhibitor. It is commonly used in research applications to study the role of dynamin in various cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Pitstop 2, by Abcam (7 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (4 mentions)
Dynole 34 2, by Abcam (3 mentions)
Dynasore, by Abcam (3 mentions)
Rolipram, by Bio-Techne (3 mentions)
Cytochalasin d, by Merck Group (3 mentions)
Monensin, by Merck Group (3 mentions)
U73122, by Merck Group (2 mentions)
Wortmannin, by Merck Group (2 mentions)
Ikarugamycin, by Merck Group (2 mentions)
Alprenolol, by Bio-Techne (2 mentions)
Isoproterenol iso, by Merck Group (2 mentions)
Isobutylmethylxanthine ibmx, by Merck Group (2 mentions)
Cgp 12177, by Bio-Techne (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Forskolin fsk, by Merck Group (2 mentions)
Hp genomewide sirna collection, by Qiagen (2 mentions)
Bafilomycin a1, by Bio-Techne (2 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
39 protocols using dyngo 4a
1
Fluorescent Protein Fusion Protocols
2
Multimodal Regulation of EGF Signaling
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EGF, genistein, genistin, fluphenazine, concanamycin A (ConA), bisindolylmaleimide (BIM) I, U73122, brefeldin A (BFA), and thapsigargin were from Sigma-Aldrich. SU6656, W7, W5, and AKT1 inhibitor VIII were from Calbiochem. Lapatinib was from LC Laboratories, 125I-EGF from Perkin Elmer, bafilomycin A1 (BafA1) from Enzo Life Sciences, Torin1 from Tocris Bioscience, Dyngo-4a was from Abcam Biochemicals, and H235SO4 was purchased from Hartmann Analytics. All other chemicals were from Sigma-Aldrich or Merck unless otherwise stated. The environment-sensitive probe NR12S was a kind gift from Prof. A. Klymchenko (University of Strasbourg, Strasbourg, France).
3
Endocytosis Inhibition Assay for Cellular Uptake
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Jurkat and Nalm-6 cells were seeded at 2 × 105 cells/mL and treated with endocytosis inhibitors. To inhibit caveolin-dependent endocytosis Nystatin (80 µM, 30 mins, Sigma-Aldrich, Australia), and Methyl-β-Cyclodextrin (MβCD) (4 mM, 40 mins, Sigma-Aldrich, Australia) were used. To inhibit clathrin-mediated endocytosis (CME): Pitstop®2 (20 µM, 10 mins, Abcam, UK) and. Dyngo®4a (30 µM, 30 mins Abcam, UK) were used. To inhibit macropinocytosis: 1 hr treatment with Wortmannin (10 µM Sigma-Aldrich, Australia) or Amiloride (25 µM, Sigma-Aldrich, Australia) was used. The cells were then exposed to the star polymers for 20 mins and thereafter washed three times with PBS before analysis by flow cytometry as described. For confocal microscopy analysis, the cells were seeded at 2 × 105 cells/mL on a FluoroDish™ (World Precision Instruments, Inc.) and imaged with Leica TCS SP8 DLS (Leica Microsystems) using the 63× oil immersion objective. Hoechst 33342 (ThermoFisher Scientific) was used for nuclei staining. All experiments were performed in triplicate.
4
Dynamin Inhibitor Regulates EGFR Localization
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To inhibit endocytosis, cells were incubated for 6 min with 0.14 mM dynamin inhibitor Dyngo-4a (Abcam), diluted in Schneider’s medium from stock solution in DMSO. For controls, equivalent concentrations of DMSO were diluted in Schneider’s medium. The effect of the dynamin inhibitor Dyngo-4a on levels of EGFRGFP was quantified in FIJI, by measuring the maximal intensities at the distal ends of filopodia during 2 min before and after drug treatment. Previous to quantification, the background of acquired images was subtracted (atrous wavelet transform, scales 1–8 minus low pass image). The GFP intensity of each filopodia was normalized to the mean of maximal intensities of all filopodia within a cell before and after treatment.
5
Modulation of Phagolysosome Dynamics
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Inhibitors and the DMSO (BioShop) vehicle control were applied after phagocytosis and maintained until fixation or the conclusion of the experiment. To increase cytosolic Ca2+, cells were incubated with 10 µM of ionomycin (Sigma-Aldrich) and 1.2 mM of CaCl2 (BioShop) 1–2 h after phagocytosis for up to 1 h. For pH neutralization of the phagolysosome, macrophages were treated with 1 µM Con A (BioShop) and 10 mM NH4Cl (BioShop) 40 min after the start of phagocytosis. To inhibit proteases, cells were incubated with a protease inhibitor cocktail (Sigma-Aldrich) 40 min after phagocytosis, which included 1.0 mM 4-(2-aminoethyl)-benzenesulfonyl fluoride, 0.8 µM aprotinin, 40.0 µM bestatin, 14.0 µM E-64, 20.0 µM leupeptin, and 15.0 µM pepstatin A. To inhibit the cytoskeleton and dynamin, macrophages were treated with the following inhibitors 40 min after phagocytosis: 10 µM nocodazole (Sigma-Aldrich), 10 µM Taxol (Sigma-Aldrich), 2 µM cytochalasin D (EMD Millipore), 1 µM jasplakinolide (EMD Millipore), 5 µM dyngo-4a (Abcam), and 5 µM dynole 34-2 (Abcam). For clathrin inhibitors, cells were incubated with 10 µM of Pitstop 2 (Abcam) or 0.5–2.0 µg/ml ikarugamycin (Sigma-Aldrich) 40 min to 4 h after the start of phagocytosis. To inhibit de novo protein synthesis, macrophages were treated with 1 µM cycloheximide (BioShop) 1 h after phagocytosis.
6
Endocytosis Inhibitor Effects on Degranulation
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Prochlorperazine (PCZ, Sigma, Munich, Germany) and Dyngo4A (Abcam, Cambridge, UK) were resuspended in 0.1% (v/v) DMSO (Sigma, Munich, Germany), which was also used as a solvent control. Degranulation and cytotoxicity assays were performed as described above, with addition of endocytosis inhibitors to the co-culture during the last hour of the assays in concentrations of 5 μM PCZ and 30 μM Dyngo4A.
7
Visualizing Receptor Internalization Dynamics
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Two days after transfection, cells were pre-treated (20 min, 37 °C) with either DMSO, Dyngo4a (30 μM, Abcam), myrD15 (10 μM, Tocris) or myrCtrl (10 μM, Tocris), incubated live with FLAG M1 antibody (Sigma F3040, dilution 1/200) (10 min, 37 °C), stimulated with Isoproterenol (10 μM, 10 min, 37 °C), rinsed 3 times with PBS + Ca2+ and fixed with paraformaldehyde (4% in PBS, 15 min, 37 °C). Fixed cells were incubated in blocking buffer (2% BSA in PBS + Ca2+) for 1 h at RT, permeabilized (0.02% Triton-X100 in blocking buffer, 15 min, RT) and incubated with AlexaFluor555 anti-mouse secondary antibody (Thermo Fisher A31570, 1/1000) in the dark for 1 h at RT. Image acquisition and analysis was conducted following the same procedure as for Transferrin uptake assay and quantification described above.
8
Microtubule Inhibitors and Actin Disruptors
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We purchased Dyngo-4a and Nocodazole from Abcam (Cambridge, United Kingdom). Cytochalasin B was purchased from Sigma Aldrich Japan (Kawasaki, Japan). The chemicals were dissolved in DMSO to provide a stock solution of 10 mM. All compounds were aliquoted and stored at −20°C until use. Cells were exposed to the drugs by adding stock solutions to the cell medium at the following concentrations: Dyngo-4a, 10 nM; Nocodazole, 30 μM; Cytochalasin B, 20 μM, Chloroquine, 10 μM.
9
Tau Uptake in HEK 293T Cells
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Tau uptake was performed as described (12 (link)). Twenty-four h before experiments, HEK 293T cells were transfected with pDEST encoding AP180 C terminus, C-terminal–tagged FCHO, dynamin ΔPH, endophilin A2 ΔAH, RhoA T19N, Cdc42 T17N, Rac1 T17N, Arf6 T27N, Rab5 S34N, and Rab7 T22N fused to enhanced GFP (EGFP) using Lipofectamine 2000 (Invitrogen). Cells were treated with 10 μg/ml transferrin conjugated to Alexa Fluor 594 (Invitrogen), followed by an acid wash (20 mm CH3COONa, pH 4.6, 150 mm NaCl, 1 mm CaCl2) for 30 s at 21 °C. CellMask (Invitrogen, 1:1000) and small molecule inhibitors were added 10 and 30 min prior to the addition of assembled tau, respectively, and were maintained in the medium throughout the experiment. The small molecule inhibitors were: 5-(N-ethyl-N-isopropyl) amiloride (EIPA, Sigma, 100 μm ), latrunculin A (Sigma, 300 nm ), wortmannin (Sigma, 25 nm ), LY294002 (Sigma, 50 μm ), Dyngo-4a (Abcam, 20 μm ), ZCL278 (Tocris, 10 μm ), chlorpromazine (Sigma, 28 μm ), and monodansylcadaverine (Sigma, 100 μm ). Fixation was with 3.6% paraformaldehyde for 10 min. 10,000 cells each from three biological replicates were analyzed by flow cytometry (Sony Eclipse or BD Biosciences LSRII).
10
Immunoblotting and Immunofluorescence Assays
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The antibodies used were: mouse anti-FLAG (M1, Sigma); rabbit anti-APPL1 (Cell Signaling Technology); rabbit anti-EEA1 (Cell Signaling Technology); mouse anti-GAPDH (Millipore); rabbit anti-p42/44 ERK and phospho-p42/44 ERK (Cell Signaling Technology); rabbit anti-phosphoserine (Millipore); goat anti-rabbit and anti-mouse AlexaFluor 488, 555, 568, and 647 (Thermo Fisher); and goat anti-rabbit and anti-mouse horseradish peroxidase (HRP) (Thermo Fisher Scientific). The inhibitors/activators used were: Dyngo-4a (Abcam) at 30 μM (45 min pre-treatment), KT5720 (Abcam) at 10 μM (15 min pre-treatment), and 8-Br-cAMP (Sigma) at 0.5 mM (15 min pre-treatment). LH and follicle-stimulating hormone (FSH) (A.F. Parlow, National Hormone and Peptide Program, Harbor-UCLA Medical Center) were used at 10 nM, and isoproterenol (ISO; Sigma) was used at 10 μM. All concentrations of ligands used (LH and FSH at 10 nM and isoproterenol at 10 μM) induce maximal cAMP responses from dose-response curves published previously (Bouvier et al., 1987 (link), Gudermann et al., 1992 (link), Alvarez et al., 1999 (link)).
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