Materials used for this study
Reagent or resource | Source | Identifier |
---|---|---|
Antibodies | ||
Anti-α-Syn antibody (clone 42) | BD | RRID: AB_398107 |
p-S129-α-Syn (clone EP1536Y) | Abcam | RRID: AB_869973 |
p-S129-α-Syn (clone D1R1R) | Cell Signaling Technology | RRID: AB_279886 |
Anti-vinculin | Sigma-Aldrich | RRID: AB_477629 |
Anti-PSD95 (clone 7E3) | Cell Signaling Technology | RRID: AB_2721262 |
Anti-GFAP | Novus Biologicals | RRID: AB_829022 |
Anti-Iba1 | Wako Chemicals | RRID: AB_839504 |
Anti-TH | EMD Millipore | RRID: AB_390204 |
Alexa Fluor 488 AffiniPure Donkey Anti-Mouse IgG (H + L) | Jackson ImmunoResearch Laboratories | RRID: AB_2340846 |
Donkey Anti-Rabbit IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 555 | Thermo Fisher Scientific | RRID: AB_162543 |
Donkey Anti-Goat IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 | Thermo Fisher Scientific | RRID: AB_2534102 |
Chemical | ||
1× protease inhibitor cocktails | GenDEPOT | Catalog #P3100–100 |
1× phosphatase inhibitor cocktails | GenDEPOT | Catalog #P3200–020 |
Laemmli sample buffer | Sigma-Aldrich | Catalog #S3401–10VL |
Amersham ECL Prime Western Blotting Detection Reagent | Cytiva | Catalog #RPN2236 |
Kit | ||
VECTASTAIN(R) ELITE(R) ABC Anti-Rabbit IgG HRP Immunodetection Kit | Vector Laboratories | Catalog #PK-6101 |
NativePage Sample kit | Thermo Fisher Scientific | Catalog #BN2008 |
Experimental models: Organisms/strain | ||
Mouse: wild type (WT: C57BL/6J) | Jackson Laboratory | Catalog #000664 |
Mouse: SncaY39E KI | This paper | N/A |
Mouse: SncaS129D KI | This paper | N/A |
CRISPR-Cas9 reagents | ||
Alt-R CRISPR-Cas9 tracrRNA | Integrated DNA Technologies | Catalog #1072532 |
Alt-R S.p. HiFi Cas9 Nuclease V3 | Integrated DNA Technologies | Catalog #1081060 |
Oligonucleotides | ||
crRNA for SncaY39E: 5′-GGCAGCTGGAAAGACAAAAG-3′ | Integrated DNA Technologies | N/A |
crRNA for SncaS129D: 5′-GGCTTATGAAATGCCTTCAG-3′ | Integrated DNA Technologies | N/A |
ssODN for SncaY39E: 5′-TTCAAAGGCCAAGGAGGGAGTTGTGGCTGCTG CTGAGAAAACCAAGCAGGGTGTGGCAGAGGCAGCTGGAAAGACGAAG GAAGGCGTGCTGGAAGTAGGTAGGTAGTGACACTGTGACTAATGAATT GGGGTGGCTGGTGTGTGGTGTCTGATTCGTGTGCATCACAGCTTCTCA GAAGAGTGACAGCTGTGTG-3′ | Integrated DNA Technologies | N/A |
ssODN for SncaS129D: 5′-GAGGAGGGGTACCCACAGGAAGGAATCCTGG AAGACATGCCTGTGGATCCTGGCAGCGAAGCCTACGAGATGCCGGAC GAAGTAAATGCCTGTATAAAGAAAACTAAGCAAAACACTTTAGGTGTTTA ATTTGGAACACATACCATCAAAACCCTGCCACTATCAGATCTCTCTCACA-3′ | Integrated DNA Technologies | N/A |
Genotyping primer for SncaY39E and WT forward: anneals at 59° 5′-CCTCC CCTTCTGCAACTCTTCTTCTG-3′ | Sigma-Aldrich | N/A |
Genotyping primer for SncaY39E reverse: anneals at 59° 5′-CTTCCAGCACG CCTTCCTTCG-3′ | Sigma-Aldrich | N/A |
Genotyping primer for WT sequence of SncaY39E KI reverse: anneals at 59° 5′-GTCACTACCTACCTACATAGAGGACTCCCTCTTTTG-3′ | Sigma-Aldrich | N/A |
Genotyping primer for SncaS129D and WT forward: anneals at 63° 5′-GCCCT CATTATTCACCACACATGCACATAGTCCAC-3′ | Sigma-Aldrich | N/A |
Genotyping primer for WT sequence of SncaS129D KI reverse: anneals at 63° 5′-CTCTGAAGGCATTTCATAAGCCTCACT-3′ | Sigma-Aldrich | N/A |
Genotyping primer for SncaS129D reverse: anneals at 63° 5′-TTCGTCCGG CATCTCGTAGGCTTCGCT-3′ | Sigma-Aldrich | N/A |
Genomic sequencing primer for SncaY39E forward: 5′-CAACAATCAAT CAACACTGTGCC-3′ | Sigma-Aldrich | N/A |
Genomic sequencing primer for SncaY39E reverse: 5′-GTCTTTGATTCATAA GCATATTCTTGG-3′ | Sigma-Aldrich | N/A |
Genomic sequencing primer for SncaS129D forward: 5′-TCATTGAAAT GCTATGTGGGTTCTGTCTAC-3′ | Sigma-Aldrich | N/A |
Genomic sequencing primer for SncaS129D reverse: 5′-ACAAAATCCAGAT AACATTCATGACAGTAA-3′ | Sigma-Aldrich | N/A |
qPCR primer for mSnca forward: 5′-GTGACAACAGTGGCTGAGAAGAC-3′ | Sigma-Aldrich | N/A |
qPCR primer for mSnca reverse: 5′-GGTACCCCTCCTCACCCTTG-3′ | Sigma-Aldrich | N/A |
qPCR primer for mGapdh forward: 5′-AGGTCGGTGTGAACGGATTTG-3′ | Sigma-Aldrich | N/A |
qPCR primer for mGapdh reverse: 5′-TGTAGACCATGTAGTTGAGGTCA-3′ | Sigma-Aldrich | N/A |