1 WB A α sin A30P 10d α sin A30P 24h α sin A30P 0h B C D E F 0h 24h 48h 10 days A11 α Oligomer OC α Fiber α syn α monomer 0h 24h 48h ID: 1042171
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1. 15 kDaIECPrePos15 kDa1WBAα-sin A30P 10dα-sin A30P 24hα-sin A30P 0hBCDEF0h24h48h10 daysA11α-OligomerOCα-Fiberα-synα-monomer0h24h48h10dThT fluorescence (a.u.)GHSupplementary Figure 1:
2. Gene AbbreviationGene NameFORWARD PRIMERREVERSE PRIMERUp or Down regulation by α-syn-O (2-folds)VIVIT restored normal phenotypeActr2ARP2 actin-related protein 2 homolog (yeast)GCTCTCTGTCCCTTTTGACAGCAAGAAAGCAAAAATTGGAActr3ARP3 actin-related protein 3 homolog (yeast)ACCCGAGATCTTTTTCCATCAGTCCCTGAACATGGTTGAAArcactivity regulated cytoskeletal-associated proteinCAGGAGAATGACACCAGGTCCAGGCACCTCCTCTCTGTAAArhgef2rho/rac guanine nucleotide exchange factor (GEF) 2TGCCTGTAACAAGAGCATCAGAAGGGAGACAGACTGCAAAArhgef25Rho guanine nucleotide exchange factor (GEF) 25TGAGGGAAAACTGACTGCTCGCTCACCTTGATGCTGTTCTArhgef6Rac/Cdc42 guanine nucleotide exchange factor (GEF) 6TTGAGCTTTGTGCTTTGTGGGTAAAGGGCTGTTGGGCATABadBCL2-associated agonist of cell deathAGTCTTTCGAGGCCTTAGGACCCCAGTTATGACAGGACAGBaxBCL2-associated X proteinTAGCAAACTGGTGCTCAAGGATGGTCACTGTCTGCCATGTBbc3BCL2 binding component 3GGGGGTCTGTGAAGAGCATAGCAGAGCACAGGATTCACAGUPNOBcl2B cell leukemia/lymphoma 2TGACTTCTCTCGTCGCTACCGAACTCAAAGAAGGCCACAABcl2A1AB cell leukemia/lymphoma 2 related protein A1aTAACTGGGGAAGGATTGTGACCCAGATCTGTCCTGTCATCDOWNYESBcl2L1BCL2-like 1CATCCCAGCTTCACATAACCGCAATCCGACTCACCAATACUPYESBcl2L10Bcl2-like 10CTTTGCAACAGCCATCTTTTATCACAAACCCAGAACTCCABcl2L11BCL2-like 11 (apoptosis facilitator)GAGTTAGGGGCTGGCTCTACGACCAAGAAAGAGCACCTCABcl2L2BCL2-like 2CAGGAACCAGGGTCAGGTTACCTGGTTTTCCCTGACAGAABcl6B cell leukemia/lymphoma 6ACCTGAGGGAAGGCAATATCGGCTGTTCAGGAACTCTTCABdnfbrain derived neurotrophic factorGGTGCAGAAAAGCAACAAGTGCACAAAAAGTTCCCAGAGABidBH3 interacting domain death agonistCTTGCTACGGAATGCAAAGATTACCCAGGGAAAGAGGATGBikBCL2-interacting killerGGGGGTCTGTGAAGAGCATAGCAGAGCACAGGATTCACAGBirc2baculoviral IAP repeat-containing 2TGCTTGCTGTATGTCCTTCAGATGCACGCCTTTAAAAGAABirc3baculoviral IAP repeat-containing 3TTTCCAACATTTGACGTGTGGTTGCTGCAGTGTTTCCTTTBirc5baculoviral IAP repeat-containing 5GGTTTTGTGGCTTTGCTCTACTGCATTAGCAGCCCTGTATBmfBCL2 modifying factorCAGGCTTCTGGTATGTGCTTTCAAACTCTGGGCTCAGTTCBmp2bone morphogenetic protein 2TTGCACACTTGCTGTCTGTTGTTCTCACGGATTGGACAACBmp4bone morphogenetic protein 4TGAGAGCTCTGCTTTTCGTTTGGTGTCCAGTAGTCGTGTGDOWNNOCalm2calmodulin 2GAAGCAGAACTGCAGGACATTAACTTCTCGCCAAGGTTTGCalm3calmodulin 3ACCAGCTGACTGAGGAACAGGCCATCATGGTCAAGAACTCCcl2chemokine (C-C motif) ligand 2AGTTTTTGTCACCAAGCTCAGGGTCAACTTCACATTCAAACdh2cadherin 2TATGTGATGACGGTCACTGCGAAAGGCCATAAGTGGGATTCdkn1Bcyclin-dependent kinase inhibitor 1BAGCGTTTCTTCATTGCCTGTCACAAAACATGCCACTTTGGCfl1cofilin 1, non-muscleGGCTCCTACTAAACGGAAGGGTCCTCACTCAGGCAAAAGACreb1cAMP responsive element binding protein 1TAGCTGTGACTTGGCATTCATTGTTCTGTTTGGGACCTGTDstndestrinGGAGTCCAGGAAAGAAGAGCTACACAGGGGATCCTTCAAAEgr1early growth response 1ACAGCAGTCCCATCTACTCGCTCCCTGTTGTTGTGGAAACFaslFas ligand (TNF superfamily, member 6)CATGGAGTGGTCCTTAATGCAAGTAGACCCACCCTGGAAGFgf2fibroblast growth factor 2GCTGCTGGCTTCTAAGTGTGGTATGGCCTTCTGTCCAGGTGria1glutamate receptor, ionotropic, AMPA1 (alpha 1)ATAAAGGGGAATGTGGAAGCGCTACGGGATTTGTAGCAGAGria2glutamate receptor, ionotropic, AMPA2 (alpha 2)CTGCGAAAGGGATAAAGTCAGTGTGTGCTCCAGGGTATTCGrin2Bglutamate receptor, ionotropic, NMDA2B (epsilon 2)CCGCAGCACTATTGAGAACAATCCATGTGTAGCCGTAGCCHrkharakiri, BCL2 interacting protein (contains only BH3 domain)AGGCTCTGGGAAGTCTGTCTACAAAAGAGCACAAGGCAACIl1Binterleukin 1 betaCCCAACTGGTACATCAGCACTCTGCTCATTCACGAAAAGGIl6interleukin 6CTACCCCAATTTCCAATGCTACCACAGTGAGGAATGTCCAItpr1inositol 1,4,5-trisphosphate receptor 1GAAACTCCTGGAGAAGCACAGAAACGCCTTCAAACTCAAAKalrnKalirin, RhoGEF kinaseGTTCATACAGGAGGGCACACGGCTTCAGGAAGGACTTAGGUPYESMap2Microtubule-associated protein 2TCCTTCTTTCCCTCGTTTCTTCTTGCCAGCAGTCACAATASupplementary Table 1:
3. Gene AbbreviationGene NameFORWARD PRIMERREVERSE PRIMERUp or Down regulation by α-syn-O (2-folds)VIVIT restored normal phenotypeMap2K4mitogen-activated protein kinase kinase 4GAAAGAGGCAGTCTGTTGGATGGCTTCTTTTCGTTTTGTCDOWNNOMcl1myeloid cell leukemia sequence 1GGGGACTCTTAAAGCTCCAGTTTGCTGAGAGGGAACTTTGUPNOMyh10Myosin, heavy polypeptide 10, non-muscleGGGACTTGAGTGAGGAGCTGGCTTTGAACCTTTTCGCTTGMyo6myosin VIGGGCTGCACTAGATTCTTCGATTTCCAAGGTGCAGGACACNeurog2neurogenin 2CTGTGGGAATTTCACCTGTCCTAAATTTCCACGCTTGCATNgfnerve growth factorAAGCTCACCTCAGTGTCTGGTTTAGTCCAGTGGGCTTCAGDOWNNONgfrnerve growth factor receptor (TNFR superfamily, member 16)AGAAGCTGCTCAATGGTGACAATGTCAGCTCTCTGGATGCNlgn1neuroligin 1CATCTCCCTGGTCTGACATCTTGTAAGGGGACCATCTTCANos1nitric oxide synthase 1, neuronalTACACACCTGGAGACCACCTGACCTGGGACCCTGTCTACTNos2nitric oxide synthase 2, inducibleCTTTGTGCGAAGTGTCAGTGCACCTGGAACAGCACTCTCTDOWNNONr4A1nuclear receptor subfamily 4, group A, member 1AAAAGCGCCAAGTACATCTGTGCTTGGGTTTTGAAGGTAGNrg1Neuregulin 1ATGGTGAAGGACCTGTCAAATTCTTGGTTTTGCAGTAGGCNrgnneurograninCGCCCAAATACTCTCAAAAAAGGCAGGTATGGGATAGGACPak1p21 protein (Cdc42/Rac)-activated kinase 1AGGAGGTGGCCATTAAACAGCCTCCAGCCAAGTATTCCATPak3p21 protein (Cdc42/Rac)-activated kinase 3CTTTTGTGGCTTGAGGAAAAGTTGGCTTGTCAAAGGCTTAPak6p21 protein (Cdc42/Rac)-activated kinase 6ACCCACAGAGAGCCTCAGAACCCCCAGCATGTGTTAGACTPak7p21 protein (Cdc42/Rac)-activated kinase 7GCTCAAGAACTCCTTGGACAGGGTTTCCTCATGTTCTCCTPfn1profilin 1GGGAAGACCTTCGTTAGCATCATCAGCAGGACTAGCGTCTPmaip1phorbol-12-myristate-13-acetate-induced protein 1AGTCGTGGAGCTAGGGAAGTACATTGCTCTGCAGTTGTCADOWNNOPmlpromyelocytic leukemiaTAAAGAAGGTGTGGCCTCTGCTGCTGGTATAGCGGTCAGTPtenphosphatase and tensin homologACAAAGGATCTCCTCCCAACTGGCGTTCTGCCTAATCTACRab3ARAB3A, member RAS oncogene familyCAGAAGGAGTCCTCAGACCACTCTTGTCGTTGCGGTAGATRgs2regulator of G-protein signaling 2CTGGTTGGCTTGTGAAGACTACACCCTCTTCTGAGCTGTGRhebRas homolog enriched in brainCAAGATGCCTCAGTCCAAGTCGCTGTGTCTACAAGCTGAARock1Rho-associated coiled-coil containing protein kinase 1GGAAAGCAAGTCAGACCTCATGTCTGCTTGTGACTTGGAASnap25synaptosomal-associated protein 25CAGGCTCTGAATCTCTCCAACATGCGTAGTTGTTGCTTTGSncasynuclein, alphaAGATTGGGGAAAACAGGAAGCCACTGTTGTCACTCCATGASncaipsynuclein, alpha interacting protein (synphilin)CAGAAGCCACTCAGTCCAGTCATTTGTTCTCCGGATTCACSncbsynuclein, betaTTCTACCTTTTCCCCCAAACGCTACATCCGAGCAGACACTSrfserum response factorAAGATAGAGCCTGGGCAAGTACGGATATACCACACGCACTSyn1synapsin IATGCTCAGCAGCACAACATATCCAGTTACCCGACACTGATDOWNYESSyn2synapsin IITCCTCTGTTCCTTTGTGCTCCTGCCAGGAGTCCTACGTTASyngap1synaptic Ras GTPase activating protein 1 homolog (rat)CTATGCACCGAACCCAATACCCCAAGTGGTATTCATGCTCSynj1Synaptojanin 1CTGGAAGTTTGCGTGTTTCTGTCTGTGAGCGTCTGGTTCTSynpo2synaptopodin 2GGACAAGATGGAAATGTTGCGTCCCATCTGGAAGGAACTTSypsynaptophysinGGAGTGGGTCGATGTGACTTTTGGCTGACTGGTCCTCTCTSyt1synaptotagmin IACTCCAACCCCTACGTCAAGCTTTCCCAATCACACAGTGGThtyrosine hydroxylaseCTTCAGAAGAGCCGTCTCAGGAATACCACAGCCTCCAATGTnftumor necrosis factorCCCACTCTGACCCCTTTACTTTTGAGTCCTTGATGGTGGTTnfrsf21tumor necrosis factor receptor superfamily, member 21CCACCACAGACACATTCTGATGGAGCTCTTTCGGATACTGTnfsf10tumor necrosis factor (ligand) superfamily, member 10ATTGGGAAGTCAGACCTGGATCATCCGTCTTTGAGAAGCATrp53transformation related protein 53AGCTTTGAGGTTCGTGTTTGGGAACATCTCGAAGCGTTTATubb4tubulin beta 4A class IVaCAGCCTCCAAGTCCTCTCACTTCCTCGTCCTTCAGCACTTDOWNYESVamp1vesicle-associated membrane protein 1AATGTCTGCTCCAGCTCAACCCAAGACCTTGTCCACATTCVamp2vesicle-associated membrane protein 2TGAGGTTCCCATCACCTCTCCTGTGGGGTTTGCTTTTGTTXiapX-linked inhibitor of apoptosisTGTGGACATCTGGTCACTTGCTTAAAGTTCGCTCCCATCADOWNNO
4. Gene AbbreviationGene NameFORWARD PRIMERREVERSE PRIMERMvpa1Genomic DNAGGAGCCCAGTGTAGAAGAGCA AGCCAGCGAACCATATCCTGA Constitutive GenesUbcubiquitin CACACAAAGCCCCTCAATCTCCCTCCTTGTCCTGGATCTTTGapdhGlyceraldehyde-3-phosphate dehydrogenaseCTGGAGAAACCTGCCAAGTATGTTGCTGTAGCCGTATTCAPgk1phosphoglycerate kinase 1GCAGATTGTTTGGAATGGTCTGCTCACATGGCTGACTTTAHprt1Hypoxanthine phosphoribosyltransferase 1GCTGACCTGCTGGATTACATTTGGGGCTGTACTGCTTAAC
5. # Conserved NFAT Biding Sites per Species GENESHomo X Mus X Chimp X Rhes X RatHomo X MusChromosome / Promoter Region in Human GenomeNFAT Responsive Gene (3000pb)Syn122chrX:47479256-47482256 NFAT Non- Responsive (6000pb)Xiap00chrX:122987662-122993662Pmaip100chr18:57561192-57567192Map2k400chr17:11918135-11924035In order to further analyze the involvement of NFAT regulation in the genes discovered to be responsive to this pathway in qPCR we made a multiple-sequence alignment analysis to detect evolutionarily conserved NFAT transcription factor binding sites. We used Mulan server together with multiTF program for comparing multiple finished-quality sequences to identify functional elements conserved over evolutionary time (Ovcharenko I Loots GG Genome Res. 2005). We conducted two comparative genome sequence analysis in five mammalian species including, humans, mouse, chimps, rhesus and rat or only in human and mouse. SYN 1 gene has at least two well conserved putative NFAT biding site in their promoter region in all five species (Species: Homo X Mouse X Chimp X Rhesus X Rat and position from the transcription start, respectively:1 V$NFAT_Q4_01 + 643-652 gtGGACActt + 631-640 gtGGACActt + 643-652 gtGGACActt + 643-652 gtGGACActt + 625-634 gtGGACActt 2 V$NFAT_Q4_01 + 953-962 atGGAGAaac + 907-916 aaGGAAActc + 951-960 atGGAGAaac + 955-964 atGGAGAaac + 873-882 aaGGAAActc). Consistently, the NFAT non-responsive genes promoters displayed no conserved putative NFAT biding site even when only human and mouse promoter were aligned. We considered promoter region for SYN 1 gene a -3000bp and for non-regulated genes a -6000bp in relation to the transcription start. The larger promoter region considered for NFAT non-regulated regions was to further demonstrate the inexistence of a conserved putative NFAT biding site in these genes. Supplementary Table 2: