Prl tk renilla luciferase reporter plasmid

The genes for nsp1β and N were cloned from of PRRSV-2 strain VR2332 and inserted into the pXJ41 expression vector as described previously [33 (link)]. The mutant plasmids nsp1β-L126A and N-ΔNLS were constructed by PCR-based site-directed mutagenesis using specific primer pairs as follows; for nsp1β-L126A, forward 5’-TGCAGCCTCCGTTGTGCGTACTTGCCAGCGAC-3’, reverse 5’-GTCGCTGGCAAGTACGCACAACGGAGGCTGCA-3’; for N-ΔNLS, forward 5’- GGCAAGGGACCGGGAAATAAGAAGAAATCC-3’, reverse 5’- GGATTTCTTCTTATTTCCCGGTCCCTTGCC-3’. PCR-based mutagenesis was performed using the QuikChange II XL Site-Directed Mutagenesis kit (Agilent, Santa Clara, CA) according to the manufacturer’s instruction. The pIFN-β-luciferase reporter plasmid was kindly provided by Stephan Ludwig (Institute of Molecular Medicine, Heinrich Heine Universtät, Düsseldorf, Germany). The pNF-κB-luciferase reporter plasmid was purchased from Stratagene Inc (La Jolla, CA). The pRL-TK Renilla luciferase reporter plasmid was purchased from Promega (Madison, WI).

Expression plasmids for the FoxO1 triple phosphorylation mutant (Addgene Plasmid 17547, deposited by Dr. Domenico Accili) and the FoxO3a triple phosphorylation mutant (Addgene Plasmid 10711, deposited by Dr. William Sellers ), have been described previously [24 (link)] and were injected and electroporated into mouse TA muscles as described by us previously [16 (link)]. The pGL4.20 luciferase reporter plasmids containing either a wildtype Cebpb promoter fragment (−516 to −1) or a mutated version which is mutated at both FoxO binding elements (FBE1 (−234 to −225) and FBE2 (−208 to −199)) were generous gifts from Dr. Akiyoshi Fukamizu, and have been described previously [25 (link)]. The pRL-TK-Renilla luciferase reporter plasmid was purchased from Promega (Madison, WI).

5-8 F and CNE-2 cells were seeded at 5 × 10³ per well in 96-well plates the day before transfection. A 683-bp promoter region (-365 to +318) of OPN was inserted into the pGL3-Basic luciferase reporter vector (Promega, USA). The cells were co-transfected with 0.1 μg of firefly luciferase reporter construct, 0.01 μg of pRL-TK Renilla luciferase reporter plasmid (Promega, USA), and the pcDNA3.1-P2Y2 vector using Lipofectamine 2000 (Invitrogen, USA). The luciferase activity was examined using a dual-luciferase reporter assay system (Promega, USA) according to the manufacturer’s instructions, and the signal was normalized to the internal Renilla control for the transfection efficiency.

HeLa, HCT116, and SW480 cells were maintained in RPMI 1640 (Hyclone, Logan, UT, USA), and HEK293T cells were maintained in DMEM (Hyclone) supplemented with 10% fetal bovine serum (FBS) (Gibco, Carlsbad, CA, USA). The TOPFlash luciferase reporter plasmid was purchased from Addgene (Watertown, MA, USA), the pRL-TK Renilla luciferase reporter plasmid was purchased from Promega (Madison, WI, USA), and the CDK11-Flag plasmid was purchased from Genscript (Nanjing, China). Wnt3a was purchased from R&D Systems (Minneapolis, MN, USA). Antibodies to the following were used: β-catenin, CDK11, and MEC-17 (Abcam, Cambridge, MA, USA); LRP6, pLRP6, Axin1, phospho-β-catenin (Ser33/Ser37/Thr41), GSK3β, acetyl-α-tubulin, histone deacetylase 6 (HDAC6), tubulin, and N-cadherin (CST, Danvers, MA, USA); Dvl2, EEA1, and LAMP1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA); Flag, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and SIRT2 (Sigma-Aldrich, St. Louis, MO, USA); and TSG101 (GeneTex, Irvine, CA, USA). In addition, normal rabbit IgG, horseradish peroxidase (HRP)-conjugated secondary mouse antibody, and HRP-conjugated secondary rabbit antibody (CST) were used.

The HA-PIAS1 and p65 genes were cloned into the pXJ41 expression vector. The nonstructural and structural genes were individually cloned in our laboratory from the FL12 infectious clone of PRRSV as the template^{80 (link)}. The coding sequences were PCR-amplified to contain ATG translation initiation and TAG termination codons. A FLAG-tag was added at the N-terminus of each gene. For membrane protein genes such as nsp2, ORF2, ORF2a, ORF5, ORF5a, and ORF6, the FLAG-tag was added to the C-terminus. The C-terminal and N-terminal deletion mutants of PIAS1 gene (pHA-PIAS1-NTD and pHA-PIAS1-CTD, respectively) were cloned into pXJ41 using the Eco RI- and Xho I-recognition sequences. The series of N gene deletion mutant are described elsewhere^{48 (link)} and were subcloned to pXJ41. The pNF-κB-luciferase reporter plasmid was purchased from Stratagene Inc (La Jolla, CA). The pRL-TK Renilla luciferase reporter plasmid was purchased from Promega (Madison, WI). The pHA-SUMO1, pHA-SUMO2, and pHA-SUMO3 plasmids were obtained from Dr. L. Flamand (Laval University, Quebec, Canada).

293T cells (2×10⁵ cells per well) were seeded into 24-well plates. The cells were transfected with 50 ng of luciferase reporter plasmids (a gift of Prof. Xiao Shaobo from HuaZhong Agricultural University) [32 (link)], together with pCAGGS-IFNλ or pCAGGS, using Lipofectamine 2000 (Thermo Scientific, Shanghai, China). In parallel, 20 ng of pRL-TK Renilla luciferase reporter plasmid (Promega, Madison, WI, USA) was transfected to normalize transfection efficiency. Twenty-four hours after transfection, the cells were infected with B2c, rB2c-IFNλ2, and rB2c-IFNλ3 for 12 h. Luciferase activity in total cell lysates was measured using a dual-specific luciferase reporter assay system (Promega, Madison, WI, USA).