|- XL10-Gold cells are resistant to tetracycline and chloramphenicol. If the mutagenized plasmid contains only the tetR or camR resistance marker, an alternative strain of competent cells must be used.
|- Use only the Dpn I enzyme provided; do not substitute with an enzyme from another source.
Plasmid vectors for expressing human OPTN and its mutants (M98K and M98K-D474N) with HA and GFP-tags have been described [, ]. HA-S177A-OPTN and HA-M98K-S177A-OPTN mutants were made by site-directed mutagenesis from HA-OPTN and HA-M98K-OPTN respectively, following the protocol described in QuikChange Site-Directed Mutagenesis Kit (Stratagene/Agilent). S177A-OPTN and M98K-S177A-OPTN were cloned in pEGFP-C3 (Clontech, 6082–1) to produce GFP-tagged OPTN mutants. cDNA of mouse was amplified by RT-PCR using RNA from RGC-5 cells and cloned in pcDNA3.1 and EGFP vectors. GFP-K38A-Tbk1 and HA-K38A-Tbk1 were made by site-directed mutagenesis from GFP-Tbk1 and HA-Tbk1 respectively. The plasmids, pDest-GFP-LC3B and mCherry-GFP-LC3B were kindly provided by Dr. Terje Johansen (University of Tromsø, Tromsø, Norway) . Adenoviral vectors expressing wild-type OPTN and its M98K, M98K-D474N and E50K mutants have been described earlier [, ]. Full paper
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