Plenti6 v5 expression vector

The PC12 cell is a cell line derived from a pheochromocytoma of the rat adrenal medulla and was purchased from Shanghai Institutes for Biological Sciences, CAS, Shanghai, China. PC12 cells that can overexpress the wild-type AS were prepared as described in our previous reports [36 (link)]. Briefly, PCR (Applied Biosystems, Waltham, MA, USA) was used to amplify the Open Reading Frame of human A53T-AS. PCR product was then cloned into a pDrive vector (Invitrogen). A53T-AS in pcDNA3.1 was subcloned in PatI-XhoI sites of a pLenti6/V5 expression vector (Invitrogen; Carlsbad, CA, USA) at the downstream of a cytomegalovirus promoter. The orientation and sequence of the construct were confirmed by restriction analysis and DNA sequencing. Then, lentiviral stocks produced by co-transfecting the optimized packaging plasmid mixture, and pLenti expressing construct into the 293T cell line, were produced according to the manufacturer’s protocols (The ViraPower^TM Lentiviral Expression System, Invitrogen), as previously described [37 (link)]. After A53T-AS in lentiviral stocks had been transduced into the PC12 cell line, the individual stably transduced colony was subsequently selected using blasticidin (Invitrogen). Finally, A53T-AS expression level was assessed by Western blot.

The pL-Oct-1Z-3FLAG and pL-Oct-1L-3FLAG (C-end) constructs were produced by inserting a copy of the human Oct-1 isoform-coding sequence into the pLenti6/V5 expression vector (Invitrogen).
The pcDNA3.1-Oct-1A-3FLAG, pcDNA3.1-Oct-1L-3FLAG, pcDNA3.1-Oct-1Z-3FLAG, and pcDNA3.1-Oct-1ΔZ -3FLAG(C-end) constructs were produced by inserting a copy of the human Oct-1 isoform-coding sequence into the pcDNA3.1 vector. Oct-1Z cDNA contains both the uORF and the Oct-1Z coding ORF, while in the Oct-1DZ, 285 bp starting from the ATG codon of the L exon were deleted, hence, it contains only the Oct-1Z ORF.