Pgem 7zf vector

Double-stranded DNA (dsDNA) templates were purchased from Integrated DNA Technologies Inc. as gBlocks at 200-ng synthesis scale. Primers including 5′ phosphorothioate modification (T*T*T*T*T*T*) or 5′ phosphorylation (/5Phos/) were purchased from Integrated DNA Technologies Inc. at 100-nmol synthesis scale with HPLC purification. Phusion High-Fidelity PCR Master Mix with HF Buffer(100 reactions/50-liter volume) and Lambda Exonuclease (1000 units) was purchased from New England Biolabs, Inc. MinElute PCR Purification Kit was purchased from QIAGEN. Nicking endonuclease Nb.BbvCI (1000 units), restriction endonucleases Eco RI (5000 units), Xho I (5000 units) and Hind III (5000 units), T7 and T3 RNA polymerases (5000 units), PCR Cloning Kit (20 reactions), NEB 10-beta and NEB stable competent E. coli were purchased from New England Biolabs, Inc. pGEM-7zf (−) vector, Pure-yield plasmid miniprep system, and the Wizard SV Gel and PCR Clean-UP System were purchased from Promega. RNA Clean and Concentrator-25 was purchased from Zymo Research.

Human C9orf72, NDUFA8, or NDUFS8 cDNA in the pGEM-7Zf(+) vector (Promega) was transcribed and translated in vitro using the TNT-coupled reticulocyte lysate system (Promega) in the presence of L-[³⁵S]methionine (PerkinElmer). Following translation, the mixture containing [³⁵S]methionine-labeled C9orf72, NDUFA8, or NDUFS8 protein was incubated with equal amounts of mitochondria that were freshly isolated from indicated cells at 25°C for 30 min. Unimported proteins were removed by adding proteinase K (10 μg/ml) at 4°C for 10 min. After adding PMSF (2 mM) to stop the digestion, mitochondria were collected by centrifugation at 10,000 g at 4°C for 10 min and solubilized in 1X SDS sample loading buffer. The imported C9orf72, NDUFA8, or NDUFS8 protein was analyzed by SDS-PAGE. To measure the CI assembly in vitro, following the mitochondrial importation of [³⁵S] NDUFS8, with or without 50 μM CCCP, and proteinase K treatment as mentioned above, the isolated mitochondria were solubilized by adding digitonin, and the intact CI was analyzed by BN-PAGE. The radioactive signals on the transferred membrane were visualized by exposure to X-ray film and quantified by Quantity One software.