Pflag cmv 5a vector

The wild-type DCTN1 coding region was PCR-amplified from a cDNA plasmid kindly provided by Dr. Farrer MJ (University of British Columbia) using the following primers (Sigma): 5′-TCAAGGGAATTCAATGGCACAGAGCAAGAGGCAC-3′ and 5′-TCAAGGGATATCAGGGAGATGAGGCGACTGTGAA-3′. The resulting fragment was inserted into the pFLAG-CMV5a vector (Sigma) using EcoRV and EcoRI. The plasmid was cut with EcoRI and KpnI, and the insert was subcloned into pAcGFP-N3 (Clontech, Mountain View, CA, USA). Mutagenesis to create the six mutated p150^glued plasmids was performed using the Quikchange Lightning site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA). The pCIneo-TDP43-FLAG plasmid was kindly provided by Dr. Koji Yamanaka (RIKEN, Brain Science Institute, Wako, Japan).

Total RNA was extracted from AZ-521 cells using ISOGEN II (NIPPON GENE, Tokyo, Japan) according to the manufacturer’s instructions. Complementary DNA (cDNA) was synthesized from 5 μg of total RNA using Primer Script II 1st strand cDNA Synthesis Kit (TaKaRa Bio, Shiga, Japan). Primers used for Bcl-xL amplification were 5′-gaattcgccaccatgtctcagagcaac;cgggagct-3′ and 5′-gtcgactttccgactgaagagtgagcccagcagaa-3′. cDNA was amplified in 25 μl of PrimeSTAR Max mixture according to the manufacturer’s protocol (TaKaRa Bio). The PCR conditions were as follows: 35 cycles of 98 °C for 10 s, 55 °C for 15 s and 72 °C for 60 s. To add 3’ adenine-overhangs, ExTaq polymerase (0.5 μl, TaKaRa Bio) was incubated with the reaction mixture at 72 °C for 10 min. PCR products were subjected to electrophoresis on 1% agarose gels containing ethidium bromide, and the band was extracted with a FastGene Gel/PCR Extraction Kit (Nippon Genetics Co. Ltd., Tokyo, Japan), subcloned into pMD20-T vector (TaKaRa Bio) and then inserted in the cloning sites (EcoR1 and Sal1) of pFLAG-CMV-5a vector (Sigma Aldrich). Cells were cultured in a 24-well plate (1×10⁵ cells per well) overnight and transfected with 0.5 μg of plasmids using X-tremeGENE HP DNA Transfection Reagent (Roche). After a 24-h incubation, cells were treated with the toxins for 10 h.