Cloning and Validation of ORFs

The open reading frame (ORF) encoding full-length DJ-1 was amplified from HEK293 cells cDNA, while the ORFs encoding the N-terminal domain of LRRK2 (amino acids 1–266, [25 (link)]) and full-length α-synuclein were amplified from pre-existing plasmids (Addgene: pDEST53-LRRK2-WT #25044 and EGFP-alpha synuclein-WT #40822, respectively). All ORFs were inserted into pFLAG-CMV-5a (Sigma-Aldrich) and pcDNA3.1(+)-PARP1cd [23 (link)] vectors. ORFs encoding α-synuclein mutants (E46K and A53T) were generated by PCR-based site-directed mutagenesis. All cloned DNA sequences were verified by DNA sequence analysis.

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Osuagwu N., Dölle C, & Tzoulis C. (2019). Poly-ADP-ribose assisted protein localization resolves that DJ-1, but not LRRK2 or α-synuclein, is localized to the mitochondrial matrix. PLoS ONE, 14(7), e0219909.

Publication 2019

pFLAG-CMV-5a

Amino acids Cdna Dna sequence analysis Dna sequences Hek293 cells Lrrk2 Orfs Plasmids Site directed mutagenesis Vectors α synuclein

Corresponding Organization : Haukeland University Hospital

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Variable analysis

independent variables

ORF encoding full-length DJ-1
ORFs encoding the N-terminal domain of LRRK2 (amino acids 1–266)
ORFs encoding full-length α-synuclein
ORFs encoding α-synuclein mutants (E46K and A53T)

dependent variables

Not explicitly mentioned

control variables

Not explicitly mentioned

controls

Positive controls: None specified
Negative controls: None specified

Annotations

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