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Psv40 rl

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The PSV40-RL is a laboratory equipment product manufactured by Promega. It is designed for a specific core function, but a detailed and unbiased description cannot be provided while maintaining the requested conciseness and lack of interpretation or extrapolation.

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Lab products found in correlation

Dual luciferase reporter assay system, by Promega (2 mentions) Pmir report reporter vector, by Thermo Fisher Scientific (1 mentions) Qiaamp viral rna mini kit, by Qiagen (1 mentions)

5 protocols using psv40 rl

1

Measuring NF-κB Transcriptional Activity

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To measure NF-κB transcriptional activity, BV2 cells or primary microglia were transfected with pGMNF-κB-Luc (GM-022001, Genomeditech). Cotransfection of Renilla luciferase under the control of the SV40 early enhancer/promoter region (pSV40-RL, Promega) was used to normalize for transfection efficiency. After treatment for 48 h, the dual-luciferase reporter assay system (Promega, USA) was used to determine NF-κB reporter activity. All transfections were performed at least two times, in triplicate.
Hou Y., Xiao X., Yu W, & Qi S. (2021). Propofol Suppresses Microglia Inflammation by Targeting TGM2/NF-κB Signaling. Journal of Immunology Research, 2021, 4754454.
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2

MiR-223 Regulation of FoxO1 3'UTR in Jurkat Cells

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To assess miR‐223 repressing the 3′ UTR of FoxO1 mRNA in Jurkat cells, 1.5 × 106 cells were transfected with 200 ng of pMIR‐REPORT™ Reporter Vector (Ambion Inc.) or 200 ng of pMIR‐REPORT‐FoxO1‐3′ UTR, 10 ng of Renilla luciferase reporter pSV40‐RL (transfection control; Promega) and 50 nM of miR‐223‐3p precursor molecules or equal amounts of miRNA precursor molecules‐negative controls. At 48 hrs after transfection, luciferase assays were performed using the Dual‐Luciferase Reporter Assay System (Promega) according to the manufacturer's instructions. All experiments were carried out five times.
Lee J., Kim C.J., Kim J., Lee D., Ahn S, & Yoon B.H. (2017). Increased miR‐223 expression in foetal organs is a signature of acute chorioamnionitis with systemic consequences. Journal of Cellular and Molecular Medicine, 22(2), 1179-1189.
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3

SARS-CoV-2 ORF6 Mutant Plasmid Construction

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The HA-tagged SARS-CoV-2 wild-type and M58R ORF6 expression plasmids are kindly provided by Prof. A. García-Sastre (CEIRS program; NIAD Centers of Excellence for Influenza Research and Surveillance; Mount Sinai School of Medicine, New York), while plasmid encoding HA-tagged SARS-CoV-2 ORF6 deleted by the last amino-acid (ORF6-∆61) mutant was prepared by standard cloning procedures using a naturally occurring virus variant (GenBank accession numbers OP002141). The ∆61 ORF6 coding sequence was amplified by Reverse-Transcription (RT)-Polymerase Chain Reaction (PCR) from viral RNA purified by using the QIAamp viral RNA mini kit (Qiagen, Milan, Italy) and cloned in the pCAGGS-MCS plasmid, in frame with a C-terminal HA-tag, at the EcoRI and XhoI unique sites. Recombinant plasmids are verified by sequencing. The pARE reporter plasmids encoding Firefly luciferase downstream of the Antioxidant Responsive Elements (ARE) and the Renilla luciferase downstream of the constitutively active SV40 promoter (pSV40-RL) are purchased from Promega (Milan, Italy). The plasmid encoding FLAG-tagged NRF2 was a gift from Randall Moon (Addgene plasmid #36971).
De Angelis M., Anichini G., Palamara A.T., Nencioni L, & Gori Savellini G. (2023). Dysregulation of intracellular redox homeostasis by the SARS-CoV-2 ORF6 protein. Virology Journal, 20, 239.
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4

STAT3 Regulation of CCL2 Promoter Activity

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293 T cells were plated at 60-70% confluence in 24-well plates and transiently transfected with 1 μg of different fragments of CCL2 promoter-connected luciferase reporter constructs together with expression plasmids for STAT3 using FuGENE 6 transfection reagent (Roche Applied Science). Empty pcDNA3.1 vector was used as a control. Firefly luciferase activity was normalized to that of Renilla luciferase, which were cotransfected under the control of the SV40 early enhancer/promoter region (pSV40-RL, Promega).
Xue J., Ge X., Zhao W., Xue L., Dai C., Lin F, & Peng W. (2019). PIPKIγ Regulates CCL2 Expression in Colorectal Cancer by Activating AKT-STAT3 Signaling. Journal of Immunology Research, 2019, 3690561.
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5

Constructing GNAQ Mutant Plasmids

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For the construction of the GNAQ mutant-expressing plasmid, the human GNAQ TrueORF Gold cDNA clone was purchased from Origene (Rockville, MD). This clone contains the full-length open reading frame of GNAQ with His-DDK tags at the 3’ end in a pCMV6-Entry vector. The tags were removed by PCR amplification using a 5’ primer outside of the ribosome binding site and the Kozak sequence while adding a BamH1 site (5’-GTCGACTGGATCCGGTACCGAGGAGATC-3’) and a 3’ primer designed to stop transcription at the correct site while adding an Mlu1 site (5’-AGGACTTACGCGTTCATTAGACCAGATTGTACTCCTTC-3’). The full-length GNAQ without the tags was directionally cloned into a pCMV6 vector through digestion with BamH1 and Mlu1. The following mutations were introduced separately into GNAQ using site-directed mutagenesis: c.548G>A (p.R183Q), c.626A>T (p.Q209L), c.626A>G (p.Q209R), and c.27C>A (p.C9X) (premature termination codon). All point mutations and the fidelity of the full-length clones were confirmed by Sanger sequencing. The reporter plasmids used for the luciferase assay were the serum response element plasmid (pSRE)–Luc (Agilent, Santa Clara, CA) and pSV40-RL (Promega, Madison, WI).
Galeffi F., Snellings D.A., Wetzel-Strong S.E., Kastelic N., Bullock J., Gallione C.J., North P.E, & Marchuk D.A. (2022). A novel somatic mutation in GNAQ in a capillary malformation provides insight into molecular pathogenesis. Angiogenesis, 25(4), 493-502.
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