Mission short hairpin rna shrna plasmids

MISSION short-hairpin RNA (shRNA) plasmids targeting hRIP3 mRNA (NM_006871), hMLKL mRNA (NM_152649), and non-targeting control sequences (NM_027088) were obtained from Sigma-Aldrich. Lentiviral plasmids were transfected into 293TN cells (System Biosciences, LV900A-1) using Lipofectamine 2000 (Invitrogen, 11668019). Pseudoviral particles were collected 2 days after the transfection and infected into cells with polybrene (8 μg/mL). Infected cells were puromycin selected two days after infection, and knockdown was confirmed by immunoblotting.

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.

MISSION short-hairpin RNA (shRNA) plasmids targeting hMLKL mRNA (NM_152649), and non-targeting control sequences (NM_027088) were obtained from Sigma-Aldrich. Lentiviral plasmids were transfected into 293T cells (System Biosciences, LV900A-1) using Lipofectamine 2000 (Invitrogen, 11668019). Pseudoviral particles were collected 48 h after the lentiviral plasmid transfection and infected into cells with polybrene (8 μg/mL). Infected cells were puromycin selected two days after infection, and knockdown was confirmed by immunoblotting.