Construction of myc-His-GCase plasmids was described elsewhere [36 (link)]. pSC2-6myc ARTS was described in [34 (link)]. MISSION shRNA plasmid (TRCN0000000285) encoding parkin-targeted shRNA was purchased from Sigma-Aldrich (St Louis, Mo, USA).
To construct the PARIS-expressing vector, a 1.9 kb PARIS cDNA fragment was amplified using Phusion high-fidelity DNA polymerase (New England Biolabs, Ipswich, USA) from the plasmid cFUGW-lenti-PARIS, a kind gift from Dr. Ted M. Dawson (Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA). The PARIS cDNA fragment was cloned into the Ecl136II site of pEGFPC3 vector plasmid (Clontech Laboratories Inc. CA, USA). Gibson assembly technology (New England Biolabs, Ipswich, USA) was used for the cloning. For knockdown of parkin, MISSION short hairpin RNA (shRNA) plasmids, encoding small interfering RNAs (siRNAs) targeting parkin, were purchased from Sigma Aldrich (St Louis, Mo, USA). Of all the existing vectors, TRCN0000000285 successfully knocked down human parkin. As a control, a pLKO.1 plasmid (Sigma Aldrich, St Louis, Mo, USA) harboring shRNA against GFP was used.
To construct the PARIS-expressing vector, a 1.9 kb PARIS cDNA fragment was amplified using Phusion high-fidelity DNA polymerase (New England Biolabs, Ipswich, USA) from the plasmid cFUGW-lenti-PARIS, a kind gift from Dr. Ted M. Dawson (Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA). The PARIS cDNA fragment was cloned into the Ecl136II site of pEGFPC3 vector plasmid (Clontech Laboratories Inc. CA, USA). Gibson assembly technology (New England Biolabs, Ipswich, USA) was used for the cloning. For knockdown of parkin, MISSION short hairpin RNA (shRNA) plasmids, encoding small interfering RNAs (siRNAs) targeting parkin, were purchased from Sigma Aldrich (St Louis, Mo, USA). Of all the existing vectors, TRCN0000000285 successfully knocked down human parkin. As a control, a pLKO.1 plasmid (Sigma Aldrich, St Louis, Mo, USA) harboring shRNA against GFP was used.
Free full text: Click here
