2010 hvj strain emcv atcc vr 1762 strain dualluciferase reporter assay system promega  (ATCC)

Journal: EMBO Reports

Article Title: Cholesterol restriction primes antiviral innate immunity via SREBP1-driven noncanonical type I IFNs

doi: 10.1038/s44319-024-00346-9

Figure Lengend Snippet: Reagents and tools table

Article Snippet: EMCV , ATCC , VR-1762 strain.

Techniques: Recombinant, Sequencing, CRISPR, Knock-Out, Cloning, Plasmid Preparation, Membrane, Protease Inhibitor, Software, Virus, Reporter Assay, Chromatin Immunoprecipitation, Magnetic Beads, Enzyme-linked Immunosorbent Assay

Journal: Nature microbiology

Article Title: Enterovirus Pathogenesis Requires the Host Methyltransferase SETD3

doi: 10.1038/s41564-019-0551-1

Figure Lengend Snippet: a, High-confidence CV-B3 2A-interacting host proteins with a MiST score > 0.75 in a comprehensive CV-B3-host protein-protein interaction map generated using affinity tag-purification mass spectrometry (AP-MS). Proteins are grouped into complexes and pathways. Highlighted in red, SETD3. b, Immunoprecipitation-western blot (IP-WB) of cells transfected with FLAG-SETD3 and CV-B3 2A-Strep. Unrelated EMCV 2A-Strep was used as control. Representative IP-WB of n = 2 biologically independent replicates showing similar results. c, AP-MS of cells expressing 2A proteins from a panel of indicated enteroviruses (7 in total). Upper panel, median MiST scores of these 7 affinity purifications. Top 200 hits are ranked along the x-axis. Cutoff median MiST score of 0.75 indicated by dotted line. Labeled and highlighted in red: NUP98, SETD3, and EIF4G1. Bottom panel, enrichment of NUP98, SETD3, and EIF4G1 intensity (over control) in affinity purifications of the individual enterovirus 2A proteins measured by targeted proteomics. Data represent the mean with SEM of n = 4 independent biological replicates, except for CV-A10, PV-1 and RV-C15 ( n = 3). d , Alanine scanning characterization of protein-protein interactions between 2A and SETD3 using the CheckMate mammalian two-hybrid system. Red, 2A mutations that disrupted 2A-SETD3 interaction (< 10-fold activation). 2-dimensional schematic and 3-dimensional structure of 2A: purple, known catalytic residues; blue, highly conserved residues that coordinate ion binding; red, putative 2A residues involved in SETD3 binding. Labeled and circled, four mutants tested in e . e, Replication kinetics of CV-B3-RLuc RNA in wild-type H1-HeLa cells. Cells were transfected with full-length wild-type virus RNA, a mutant that harbored the 2A C107A catalytic mutation, or mutants that carried the indicated 2A mutations (failed to interact with SETD3) without the C107A catalytic mutation. For panels d and e , data represent the mean with SEM of n = 3 independent biological replicates. P -values calculated by either ordinary one-way ANOVA ( d ) or two-way ANOVA with Holm-Sidak correction on log-transformed data ( e ). For panel d , all mutants with markedly disrupted SETD3 interaction (< 10-fold activation; red) have a statistically significant reduction in activation compared to 2A C107A control ( P < 0.0001). For panel e , at 4 hpi and beyond, the differences observed between WT and all other mutants are statistically significant ( P < 0.0001).

Article Snippet: Encephalomyocarditis virus (EMCV) Mengovirus strain vMwt (VR-1598) was purchased from ATCC and was propagated and titered by plaque assay on H1-HeLa cells.

Techniques: Generated, Purification, Mass Spectrometry, Protein-Protein interactions, Immunoprecipitation, Western Blot, Transfection, Control, Expressing, Labeling, Targeted Proteomics, Activation Assay, Binding Assay, Virus, Mutagenesis, Transformation Assay