EZ spheres were differentiated into dopaminergic neurons using fibroblast growth factor-8, purmorphamine, and growth factors as described previously (Ebert et al., 2013 (link)). Telencephalic excitatory projection neurons were derived from EZ spheres as described previously (Ebert et al., 2013 (link)). Sensory neurons were derived from monolayer iPSCs as described previously (Chambers et al., 2012 (link)). For LRRK2 kinase inhibition experiments, 1 μM GSK2578215A (Tocris) was added at every feeding starting at 1 week prior to endpoint analysis. DMSO (1 μM) was used as the vehicle control. All analyses were performed at 5 weeks of total differentiation for dopaminergic neurons and 4 weeks of total differentiation for glutamatergic and sensory neurons.
Gsk2578215a
Manufactured by Bio-Techne
Sourced in United Kingdom, United States
GSK2578215A is a small molecule inhibitor that selectively inhibits the kinase activity of TANK-binding kinase 1 (TBK1) and inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase epsilon (IKKε). It is commonly used in research applications to study the role of these kinases in cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Lrrk2 in 1, by Bio-Techne (5 mentions)
Lrrk2 in 1, by Merck Group (2 mentions)
Nocodazole, by Merck Group (2 mentions)
Czc25146, by Bio-Techne (2 mentions)
Mli 2, by Bio-Techne (2 mentions)
Mg132, by Merck Group (2 mentions)
Mli 2, by Merck Group (1 mentions)
Bafilomycin a1, by Merck Group (1 mentions)
Lipofectamine ltx, by Thermo Fisher Scientific (1 mentions)
Gentamicin, by Thermo Fisher Scientific (1 mentions)
Anti acetylated tubulin, by Merck Group (1 mentions)
Pdsred2 mito vector, by Takara Bio (1 mentions)
Anti 4 hydroxynonenal, by Abcam (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Dmem f12, by Thermo Fisher Scientific (1 mentions)
Mdivi 1, by Merck Group (1 mentions)
Anti p62, by BD (1 mentions)
Lipod293, by SignaGen (1 mentions)
15 protocols using gsk2578215a
1
Differentiation of Stem Cells into Neurons
2
Obtaining and Characterizing Small Molecule Inhibitors
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GSK2578215A (Reith et al, 2012 ) was obtained from Tocris or GlaxoSmithKline; MLI‐2 (Fell et al, 2015 ) was obtained from Merck; Bafilomycin A1 (#B1793) was obtained from Sigma, and HG‐10‐102‐01 (Choi et al, 2012 ), LRRK2‐IN1 (Deng et al, 2011 ) and Vps34‐IN1 (Bago et al, 2014 ) were custom synthesised by Natalia Shapiro (University of Dundee, UK).
3
Differentiation and Manipulation of Human and Rat Neuronal Cells
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Human SH-SY5Y cells were cultured in DMEM containing 10 % fetal bovine serum at 37 °C with 5 % CO2 and differentiated in medium containing all-trans retinoic acid (10 μM) for 6–7 days to obtain dopaminergic neuron-like properties.
Rat primary cortical neuron cultures were carried out as described previously [31 (link)]. The LRRK2 specific kinase inhibitors LRRK2 IN-1 (438193, Merck Millipore, Darmstadt, Germany) and GSK2578215A (4629, Tocris Biosciences, Bristol, United Kingdom) was added as indicated. Transfection of plasmid was carried out with Lipofectamine LTX (Invitrogen) as recommended by the manufacturer.
Rat primary cortical neuron cultures were carried out as described previously [31 (link)]. The LRRK2 specific kinase inhibitors LRRK2 IN-1 (438193, Merck Millipore, Darmstadt, Germany) and GSK2578215A (4629, Tocris Biosciences, Bristol, United Kingdom) was added as indicated. Transfection of plasmid was carried out with Lipofectamine LTX (Invitrogen) as recommended by the manufacturer.
4
Mitophagy and Neurodegeneration Protocol
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DMEM-F12 (Dulbecco's modified Eagle's medium), penicillin–streptomycin, gentamicin and fetal bovine serum (FBS) were purchased from Gibco-Invitrogen (Carlsbad, CA, USA). GSK 2578215A and LRRK2-IN-1 were purchased from Tocris Bioscience (Minneapolis, MN, USA). Stealth RNAi duplexes were purchased from Life Technologies (Carlsbad, CA, USA). BCA protein assay was from Pierce (Rockford, IL, USA). The pDsRed2-mito vector was provided by Clontech (BD Biosciences, San Jose, CA, USA), GFP-LC3 was provided by Dr. JM Fuentes (Universidad de Extremadura, Badajoz, Spain), Drp1-GFP was provided by T Wilson and Dr. S Strack (Department of Pharmacology, University of Iowa Carver College of Medicine, Iowa City, IA, USA) and mRFP-GFP-LC3 was provided by Dr. E Kneck (Laboratory of Cellular Biology, Centro de Investigación Príncipe Felipe, Valencia, Spain). Anti-4 hydroxynonenal and LRRK2 antibodies were purchased from Abcam (Cambridge, UK); anti-p62 from BD (San Jose, CA, USA) and Alexa Fluor 488 were from Molecular Probes (Carlsbad, CA, USA), Invitrogen (Carlsbad, CA, USA); and anti-acetylated tubulin was from Sigma-Aldrich (St. Louis, MO, USA). The TUNEL method (MEBSTAIN Apoptosis Kit) was purchased from MBL (Carlsbad, CA, USA). Mdivi-1 was purchased from Sigma-Aldrich.
5
Dissolving IN-1 and GSK2578215A
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IN‐1 and GSK2578215A were purchased from Tocris Bioscience (Bristol, UK). Both compounds were dissolved in DMSO to 10 mmol L−1, then diluted with Krebs at the tested concentrations.
6
LRRK2 and RAB Transfection in HEK293T and SH-SY5Y Cells
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HEK293T cells were cultured as described (Madero-Perez et al., 2018 (link)) and transfected at 80% confluence with 2 μg of LRRK2 constructs and 200 ng of RAB constructs where indicated, and 6 μl of LipoD293 (SignaGen Laboratories) per well of a 6-well plate for 5 h in full medium, resulting in a roughly 30% transfection/co-transfection efficiency, respectively. Cells were split 1:5 the following day, and processed for immunocytochemistry or Western blotting 48 h after transfection.
SH-SY5Y cells stably expressing GFP, flag-tagged wildtype LRRK2, or flag-tagged G2019S-mutant LRRK2 were cultured as described (Madero-Perez et al., 2018 (link)) and subcultured at a ratio of 1:6 twice a week. Transfection of cells was carried out at 80% confluence with 0.4 μg of DNA and 1.5 μl of Lipofectamine 2000 (Invitrogen) per well of a 24-well plate in 200 μl OptiMEM. Five hours later, cells were changed into full medium, passaged the following day at a 1:5 ratio onto coverslips, and fixed and stained 72 h after transfection.
Where indicated, cells were treated with brefeldin A (7.5 μg/ml, Sigma-Aldrich) or with nocodazole (200 nM, Sigma-Aldrich) for 3 h, or with 100 nM MLi2 (MRC PPU, Dundee, United Kingdom), 500 nM LRRK2-IN1 (obtained through the MJFF) or 500 nM GSK2578215A (Tocris) for 1 h before fixation.
SH-SY5Y cells stably expressing GFP, flag-tagged wildtype LRRK2, or flag-tagged G2019S-mutant LRRK2 were cultured as described (Madero-Perez et al., 2018 (link)) and subcultured at a ratio of 1:6 twice a week. Transfection of cells was carried out at 80% confluence with 0.4 μg of DNA and 1.5 μl of Lipofectamine 2000 (Invitrogen) per well of a 24-well plate in 200 μl OptiMEM. Five hours later, cells were changed into full medium, passaged the following day at a 1:5 ratio onto coverslips, and fixed and stained 72 h after transfection.
Where indicated, cells were treated with brefeldin A (7.5 μg/ml, Sigma-Aldrich) or with nocodazole (200 nM, Sigma-Aldrich) for 3 h, or with 100 nM MLi2 (MRC PPU, Dundee, United Kingdom), 500 nM LRRK2-IN1 (obtained through the MJFF) or 500 nM GSK2578215A (Tocris) for 1 h before fixation.
7
Reagents for Immune Cell Stimulation
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The following antibodies were used for cell stimulation: purified anti-mouse CD38 (Clone 90; BioLegend, 102702), purified rat IgG2a, κ isotype control (Clone RTK2758; BioLegend, 400565). In certain experiments, the following reagents/inhibitors were used at the indicated concentration in cell culture: LPS (E. Coli Serotype R515; Enzo Life Sciences, ALX-581-007-L001), NAADP-AM (3 μM; AAT Bioquest, 21000), GSK2578215A (LRRK2 inhibitor, 1 µM; Tocris Bioscience, 4629), GPN (200 µM; Santa Cruz Biotechnology, sc-252858), EDTA (2 mM; Sigma-Aldrich, E6758), thapsigargin (500 nM; Tocris Bioscience, 1138), 8-Br-cADPR (100 µM; BIOLOG Life Science Institute, B065), 8-Br-ADPR (100 µM; BIOLOG Life Science Institute, B051), Ned-19 (100 µM; Cayman Chemical, 17527), cyclosporine (10 µM; R&D Systems, 1101/100), FK506 (5 nM; Eton Bioscience Inc, 1100060052), CHIR 99021 (GSK3B inhibitor, 5 µM; Tocris Bioscience, 4423), BAPTA-AM (10 µM; R&D Systems, 2787), EGTA-AM (10 µM; AnaSpec Inc, AS-84100), GSK2606414 (EIF2AK3/PERK inhibitor, 10 µM; Tocris Bioscience, 5107), and dorsomorphin (5 µM; Tocris Bioscience, 3093).
8
Inhibitors of Microtubule Dynamics
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Trichostatin A, tubacin and nocodazole were from Sigma Aldrich, PTL from Eurodiagnostico, LRRK2-IN1, TAE684 and CZC25146 from the Michael J. Fox Foundation, GSK2578215A from Tocris, compound 68 (ID 9108605) and compound 70 (ID 9119202) from Chembridge Corporation (San Diego, USA), and GNE-0877 and GNE-7915 from MedchemExpress (USA), and MLi2 from MRC PPU, Dundee, UK. Natural streptolysin-O was from Abcam (ab63978), and non-hydrolyzable GTP test kit from Jena Bioscience (NK-102).
9
LRRK2 inhibitor-mediated autophagy regulation
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Chemical compounds were purchased as follows: LRRK2in1 [24 (link)] from the Division of Signal Transduction Therapy, School of Life Sciences, University of Dundee, U.K.; GSK2578215A and MLi-2 from Tocris [25 (link)]; bafilomycin-A1 (B1793-2UG) and cycloheximide (01810-1G) from Sigma-Aldrich; torin-1 (CAY10997) from Cayman Chemicals. Antibodies used were as follows: LC3 (NB100-2220, Novus Biologicals); total P70S6K (sc-8418, Santa Cruz); phospho Thr389 P70S6K (sc-11759, Santa Cruz); p62 (610833, BD Transduction Labs); total ULK1 antibody (8054 and 4773, Cell Signalling); phospho Ser757 ULK1 antibody (6888 and D7O6UCell Signalling); phospho Ser555 ULK1 antibody (5869, Cell Signalling); total LRRK2 antibody (MJFF2, Abcam); phosphor Ser935 LRRK2 antibody (UDD2, Abcam) and β-actin (A1978, Sigma-Aldrich). AMPK activator (A769662) was kindly provided by Dr MPMS.
10
Stimulating Cell Responses with Diverse Agents
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Where indicated, cells were treated with recombinant human IFN-γ (R&D Systems), LPS (LPS from E. coli O111:B4), poly(I:C) (both Invivogen), ionomycin, MG132, colchicine, PMA (all from Sigma-Aldrich), LRRK2-IN-1 and GSK2578215A (both Tocris), Mli-2 (Abcam), MCV1 (HPVIVIT, Calbiochem), recombinant human NRG1 (BioLegend), and PTX (STEMCELL Technologies).
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