Plvx ires zsgreen

CaCl₂ was purchased from Bodi Chemical Co., Ltd. (Tianjin, China). Antibodies specific against NeuN and Alexa Fluor-488, Alexa Fluor-555, and HRP-labeled secondary antibodies were purchased from Cell Signaling Technology (Danvers, MA, United States). S(+)-Ketamine (60 mg/kg, for 1 h), SC-51322 (30 nM, for 12 h), PF-04418948 (100 nM, for 12 h), and DG-041 (60 nM, for 12 h) were obtained from R&D Systems (Minneapolis, MN, United States), and CJ-42794 (40 nM, for 12 h) was obtained from MedChem Express (Monmouth Junction, NJ, United States). High-fidelity (HF) restriction enzymes for EcoRI, BamHI, XhoI, and AgeI were purchased from New England Biolabs (Beverly, MA, United States). DAPI was procured from Beyotime Institute of Biotechnology (Haimen, China). The plko.1-puro, psPAX₂, pMD₂.G, and plvx-IRES-zsgreen vectors were purchased from Addgene (Sidney, SD, United States). All the reagents used for the quantitative (q)RT-PCR and SDS-PAGE experiments were purchased from Bio-Rad Laboratories (Hercules, CA, United States), and all other reagents were obtained from Invitrogen (Carlsbad, CA, United States), unless otherwise specified.

Human embryonic kidney 293T (HEK-293T, ATCC CRL-11268) cells, rabbit kidney cells (RK13, ATCC CCL-37), and Vero cells (ATCC CCL-81) were maintained in DMEM (Gibco, USA) supplemented with 10% (v/v) fetal bovine serum (Excell, Australia). The PRV JX-2012 BAC plasmid was as described in our previous report [19 (link)]. pDC315-EGFP is an EGFP expression plasmid described in our previous studies [20 (link),21 (link)]. The pLVX-IRES-ZSGreen (Addgene, USA) or HIV-1-based luciferase (HIV-Luc) reporter plasmid [20 (link),22 (link)] was cotransfected with the helper plasmids psPAX2 and pMD2.G (Addgene, USA). The MbPylRS/tRNA_CUA plasmid was described in our recent study [17 (link)].
tRNA gene sequences were obtained from the tRNA database (http://gtrnadb.ucsc.edu/index.html) [18 (link)]. Three ACE-tRNA (ACE-Arg^UGA, ACE-Gly^UGA and ACE-Trp^UGA) expression plasmids were synthesized directly by Sangon Biotech Company (Shanghai, China) and cloned into the pUC57 vector. These ACE-tRNAs contain 4 tandem tRNAs driven by different eukaryotic promotors.