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4, 2 mM DTT] and incubating the mixture at 30 °C for 10 min. When indicated, 1 μg of MBP (myelin basic protein) was added as general kinase substrate. To test the compounds' inhibitory effects on LRRK2 kinase activity, each chemical was prepared at 10 mM stock in dimethyl sulfoxide (DMSO) and added to the reaction mixture at the indicated concentration. Then, the whole mixture was subjected to the protein gel electrophoresis, and levels of the LRRK2 autophosphorylation or MBP phosphorylation were analyzed by an image analyzer (Typhoon 9200, GE healthcare).
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17
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79955561656
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note
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50s were calculated from the formula.
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The kinas profiling test was performed by Milipore Co. The detail information can be found in its website, www.millipore.com/drugdiscovery/ KinaseProfiler.
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27 and relative cell survival rates were calculated. To test compounds' inhibitory activities, the indicated concentration of chemicals or an equal volume of dimethyl sulfoxide (DMSO) were treated at 1 h before the treatment of hydrogen peroxide.
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26
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79955551844
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note
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To construct a homology model structure of the LRRK2 kinase domain, we used a crystal structure of transforming growth factor-beta (TGF-β) activated kinase 1 (TAK1) (PDB accession code: 2EVA ). The sequence alignment of LRRK2 and template proteins was generated using the FASTA program (http://www.ebi.ac.uk/Tools/fasta33 ). A 3D model structure of LRRK2 was built by using the HOMOLOGY module of Insight II program (Accelrys Software Inc., San Diego, USA) and was further refined by using the Discover 2.98 of the Insight II.
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