To inhibit LRRK2 kinase activity in transfected HEK293 cells, the LRRK2 kinase inhibitor LRRK2-IN-1 (Merck; Deng et al., 2011 (link)) was added to the cell culture medium 2 h before electrophysiological recordings at a final concentration of 1 μM. LRRK2-IN-1 was also added to the external bath solution at the same concentration for recordings from these pre-treated cells. The internal patch pipette solution for both HEK293 and PC12 electrophysiological recordings contained 120 mM , 10 mM CsCl, 10 mM HEPES, 4 mM Mg-ATP, 3 mM Tris-GTP, 2 mM MgCl2 and 1 mM EGTA (pH adjusted to 7.2 with CsOH). HEK293 cells were bathed in an external solution containing 145 mM tetraethylammonium chloride, 10 mM HEPES and 5 mM CaCl2. The external solution for PC12 recordings contained 115 mM NaCl, 4 mM KCl, 1 mM MgCl2, 10 BaCl2, 10 mM tetraethylammonium chloride, 10 mM glucose and 10 mM HEPES. TTX was added to this solution immediately prior to recordings at a final concentration of 1 μM to block endogenous voltage-gated Na+ currents.
Lrrk2 in 1
Manufactured by Merck Group
Sourced in Germany
LRRK2-IN-1 is a selective, potent, and ATP-competitive inhibitor of the LRRK2 kinase. It is commonly used in research to investigate the role of LRRK2 in cellular processes and its potential implications in various diseases.
Automatically generated - may contain errors
Lab products found in correlation
Gsk2578215a, by Bio-Techne (2 mentions)
Egta am, by Merck Group (1 mentions)
Bapta am, by Merck Group (1 mentions)
Lipofectamine ltx, by Thermo Fisher Scientific (1 mentions)
Anti tyrosine hydroxylase, by Merck Group (1 mentions)
Anti 4e bp1, by Cell Signaling Technology (1 mentions)
Anti actin, by Merck Group (1 mentions)
Mli 2, by Bio-Techne (1 mentions)
Mjf r21, by Abcam (1 mentions)
C41 2, by Abcam (1 mentions)
4 protocols using lrrk2 in 1
1
LRRK2 Kinase Inhibition in HEK293 Cells
2
Pharmacological Inhibition of LRRK2, Rab7, and PIKfyve
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All drugs used in this study were dissolved in DMSO, diluted into culture medium and the medium sterile filtered prior to use. The LRRK2 kinase inhibitors LRRK2-In1 and GSK2578215A were from Merck and R&D Systems, respectively. The Rab7 GTPase inhibitor, CID 1067700 (Agola et al., 2012 (link)) was from EMD Millipore. The NAADP antagonist trans-Ned-19 was synthesised as described previously (Naylor et al., 2009). Ned-K is an analogue of Ned-19 in which the fluoride has been replaced with a cyano group. Its synthesis will be described elsewhere. Both Ned-19 and Ned-K were kind gifts from A. Ganesan (School of Pharmacy, University of East Anglia, UK), Raj Gossain (School of Chemistry, University of Southampton, UK) and Sean M. Davidson (Hatter Institute, UCL, UK). The PIKfyve inhibitor, YM-201636 was from Cambridge Bioscience. BAPTA-AM and EGTA-AM were from Sigma.
3
Differentiation and Manipulation of Human and Rat Neuronal Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
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
Antibodies and Mouse Model for LRRK2
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Antibodies for LRRK2 [MJFF2 (C41-2)] (# ab133474), phospho-S935-LRRK2 [UDD2 10 ( 12)] (#ab133450), phospho-T73-Rab10 [MJF-R21] (# ab230261), Rab10 [MJF-R23] (# ab237703), and RPL10a (# ab55544) were from Abcam. Anti-tyrosine hydroxylase was from Millipore (#AB1542). Antibodies for eIF2B5(sc-28854), eIF2α (sc-11386), phospho-eIF2α-S52 (sc-101670), eIF4G3 (sc-100732), and Ndufs3 (sc-292169) were from Santa Cruz Biotechnology. Anti-Rab8 (#R66320) and Rab4 (#R68520) were from BD Transduction Laboratories. Anti-4E-BP1 (# 9452), eEF2 (#2332) and phospho-eEF2(T57) (#2331) were from Cell Signaling Technology and anti-actin (# A3853) was from Sigma-Aldrich. LRRK2-IN1 was from Merck Millipore (# 438193), GSK-2578215A (#4629) and MLi-2 (# 5756) were from Tocris Bioscience. Miglyol 812N was from Cemer Oleo, GmbH, and Co, KG (Germany). Patient cells were from the National Institute of Neurological Disorders and Stroke (NINDS) repository at the Coriell Institute for Medical Research and the Cell Line and DNA Biobank from Patients Affected by Genetic Diseases, Telethon Network of Genetic Biobanks (TNGB) (project no. GTB12001). 23 C57BL/6-Lrrk2 tm1.1Mjff /J mice developed by Michael J. Fox Foundation 24 were obtained from The Jackson Laboratory (Stock no. 016121).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!