PMC:4608092 / 9101-11056 JSONTXT

{"project":"2_test","denotations":[{"id":"26126547-23850618-55294624","span":{"begin":1743,"end":1745},"obj":"23850618"}],"text":"Plasmid Construction for RNF213 WT, RNF213 R4810K, RNF213 WEQ, and RNF213 Deletion of AAA+ and Transfection\nTo obtain RNF213 cDNA, reverse-transcriptase polymerase chain reaction (RT-PCR) was performed using 3 sets of primers (Primer N1 to N2, M1 to M2, and C1 to C2), as shown in Table S1. We used RevTra Ace reverse transcriptase and KOD FX DNA polymerase (TOYOBO, Osaka, Japan). The 3 amplified fragments were digested with restriction enzymes (NcoI and SalI, SalI and HindIII, and HindIII and NotI, respectively). Fragments were cloned between NcoI and NotI sites of pENTR4 (Thermo Scientific, Waltham, MA) to construct full-length cDNA of RNF213. The R4810K mutation and Walker B mutation (E2488Q:WEQ) were introduced by PCR-based, site-directed mutagenesis using mutated primers using Pfu Turbo DNA polymerase (Agilent Technologies, Santa Clara, CA). These mutated primers (Primers 1 to 4) were shown in Table S1. The deleted mutation (delta AAA) was introduced by the SalI site at the 8310-nucleotide position from the start codon (primer delatSalI: AAT GTC GAC GTG ATC ACA GAA GTC CTC TGC and primer M2) by PCR using KOD FX DNA polymerase and combined with RNF213 cDNA. These entry clones were used for the LR reaction (Thermo Scientific) using a destination vector with an amino-terminal 3xFLAG tagged or enhanced green fluorescent protein (EGFP) sequence under a tetracycline-regulated cytomegalovirus promoter. The design of the tagged-RNF213 WT and mutant vectors that were used in the present study are shown in Figure1. The generated constructs were confirmed by sequencing. The plasmids were transfected into cells using an Amaxa Nucleofector Device (Lonza), following the manufacturer’s recommendations, as previously reported.14\nFigure 1 Design of tag protein (FLAG or EGFP)-RNF213 WT, RNF213 R4810K, RNF213 WEQ, and RNF213 ΔAAA vector constructs. EGFP indicates enhanced green fluorescent protein; WEQ, Walker B motif; WT, wild type.\n\nC"}