recombinant human ogg1 protein  (MedChemExpress)

Journal: PLOS Pathogens

Article Title: 8-Oxoguanine DNA Glycosylase1 conceals oxidized guanine in nucleoprotein-associated RNA of respiratory syncytial virus

doi: 10.1371/journal.ppat.1012616

Figure Lengend Snippet: (A) OGG1 binds RNA containing 8-oxoGua. The secondary structure of the undenatured single-strand probe was predicted using software as described in Materials and Methods. The probe contains a single 8-oxoGua (marked in red) at the end of GGGG. EMSA was performed using single-stranded RNA (8oxo-rG), either denatured at 95°C for 5 min (Lanes 1–4) or undenatured (Lanes 5–8). Additionally, EMSA was conducted with the double-stranded RNA probe (8oxo-rG: rC), where 8oxo-rG anneals with its complementary genome sequence (rC) (Lanes 9–12). (B) Representative EMSA image demonstrating OGG1’s binding affinity for annealed RNA containing 8-oxoGua. (C) Representative image showing OGG1’s excision activity on 8-oxoGua within DNA, contrasting with its inactivity towards RNA. (D) EMSA visualization indicating that TH5487 impedes OGG1’s binding to RNA harboring 8-oxoGua. (E) EMSA depiction showing OGG1’s interaction with a DNA-RNA hybrid that includes the gene-start (GS) motif. Total RNA (1 μg) extracted from virocells (MOI = 1, 24 hpi) was hybridized with Cy5-labelled DNA probes (20 nM) containing the intergenic region (IG), the gene-start (GS), and the gene-end (GE) of G gene. Following incubation with recombinant OGG1 (100 nM), the assay proceeded to EMSA. Oligo sequences for EMSA and OGG1 glycosylase activity are listed in . n = 3 biological replicates.

Article Snippet: Specifically, GST-tagged OGG1 (TP720109, OriGene, Rockville, MD) was immobilized onto Glutathione Agarose, and subsequently incubated with varying concentrations of His-tagged nucleoprotein (NP-424V, Creative BioMart, Shirley, NY), or His-tagged M2-1 protein (purified locally).

Techniques: Software, Sequencing, Binding Assay, Activity Assay, Incubation, Recombinant

Journal: PLOS Pathogens

Article Title: 8-Oxoguanine DNA Glycosylase1 conceals oxidized guanine in nucleoprotein-associated RNA of respiratory syncytial virus

doi: 10.1371/journal.ppat.1012616

Figure Lengend Snippet: (A) Left panel: Strategy for enrichment of viral RNAs interacting with OGG1 and N protein from virocells after formaldehyde crosslinking. Treatment with TH5487 (10 μM) was initiated post inoculum (MOI = 1, 24 hpi). RNA-immunoprecipitation (RIP) was carried out using antibodies against OGG1 or RSV N protein. Right panel: Quantification of RSV genomic RNA levels after antibody pull-down, determined by qRT-PCR. Fold change was calculated by 2^- [Ct (test Ab)-Ct (negative Ab)]. n = 3 biological replicates. Statistical analysis was conducted using an unpaired Student’s t test, with results shown as means ± SD. *** p <0.001. (B) OGG1 RIP-Seq analysis in virocells (MOI = 1, 24 hpi) illustrating the distribution of RSV-specific reads across two independent experiments (visualized in blue and red tracks). Data are presented as the log2 ratio of reads obtained from anti-OGG1 immunoprecipitation relative to input RNA, mapped against the RSV genome (GenBank: M74568.1). Peaks were called positive if the log2 enrichment score was ≥1. (C) Annotation and categorization of cellular RNA reads obtained from OGG1 RIP-Seq aligned to the human genome HG38 (GenBank: GCA_000001405.29), presented as the percentage of OGG1-RIP peaks. UTR, untranslated region of mRNAs; CDS, exon regions that code for protein; und., undefined. Fig 4A created with Biorender.com .

Article Snippet: Specifically, GST-tagged OGG1 (TP720109, OriGene, Rockville, MD) was immobilized onto Glutathione Agarose, and subsequently incubated with varying concentrations of His-tagged nucleoprotein (NP-424V, Creative BioMart, Shirley, NY), or His-tagged M2-1 protein (purified locally).

Techniques: RNA Immunoprecipitation, Quantitative RT-PCR, Immunoprecipitation

Journal: PLOS Pathogens

Article Title: 8-Oxoguanine DNA Glycosylase1 conceals oxidized guanine in nucleoprotein-associated RNA of respiratory syncytial virus

doi: 10.1371/journal.ppat.1012616

Figure Lengend Snippet: (A) LC_MS/MS identification of RSV proteins co-immunoprecipitated with OGG1 (RSV MOI = 1, 24 hpi). The analysis was conducted on complexes isolated using an anti-OGG1 antibody (for endogenous OGG1) and anti-FLAG antibody (for transgenically expressed OGG1). Proteins are listed according to the number of significant peptides identified. ND not detected. (B) The entire amino acid sequence of the RSV N protein was visualized, with residues identified by LC_MS/MS highlighted in red. (C) The peptides identified by LC_MS/MS in red were visualized within the three-dimensional structure of the N protein, with RNA depicted in green. NTD, N terminal domain. CTD, C terminal domain.

Article Snippet: Specifically, GST-tagged OGG1 (TP720109, OriGene, Rockville, MD) was immobilized onto Glutathione Agarose, and subsequently incubated with varying concentrations of His-tagged nucleoprotein (NP-424V, Creative BioMart, Shirley, NY), or His-tagged M2-1 protein (purified locally).

Techniques: Liquid Chromatography with Mass Spectroscopy, Immunoprecipitation, Isolation, Sequencing

Journal: PLOS Pathogens

Article Title: 8-Oxoguanine DNA Glycosylase1 conceals oxidized guanine in nucleoprotein-associated RNA of respiratory syncytial virus

doi: 10.1371/journal.ppat.1012616

Figure Lengend Snippet: (A) Immunoblot (IB) analysis of co-immunoprecipitated extracts from Flag-OGG1 expressing hSAECs infected with RSV (MOI = 1, 24 hpi). Co-immunoprecipitation was performed using an anti-FLAG antibody or control IgG, and the blots were subsequently probed with an anti-RSV antibody. Anti-Actin immunoblotting from whole cell lysates served as a loading control. An arrow indicates the N protein. (B) Immuno-fluorescence staining of OGG1 and RSV N protein. hSAECs were mock or RSV-infected (MOI = 1), and cells were fixed at 18 and 24 hpi. The co-localization measurement between fluorophores (Alexa 594 vs. Alexa 488) was assessed using the intensity of individual fluorophore pixels, calculated as the Pearson correlation coefficient (R). Scale bars: 20 μm. (C) Proximity ligation assay (PLA) shows OGG1 and N interactions (MOI = 1, 24 hpi). PLA signals were absent when control IgG were applied. Scale bars, 50 μm. (D-E) GST pull-down assays demonstrating the interaction between OGG1 and the N protein. Immobilized GST-tagged OGG1 (100 nM) was incubated with increasing concentrations of His-tagged N, followed by washing, SDS-PAGE and immunoblotting using an anti-N antibody. (D) In a reciprocal setup, His-tagged N protein (100 nM) was incubated with varying concentrations of GST-OGG1, followed by immunoblotting with an anti-OGG1 antibody (E).

Article Snippet: Specifically, GST-tagged OGG1 (TP720109, OriGene, Rockville, MD) was immobilized onto Glutathione Agarose, and subsequently incubated with varying concentrations of His-tagged nucleoprotein (NP-424V, Creative BioMart, Shirley, NY), or His-tagged M2-1 protein (purified locally).

Techniques: Western Blot, Immunoprecipitation, Expressing, Infection, Control, Fluorescence, Staining, Proximity Ligation Assay, Incubation, SDS Page

Journal: PLOS Pathogens

Article Title: 8-Oxoguanine DNA Glycosylase1 conceals oxidized guanine in nucleoprotein-associated RNA of respiratory syncytial virus

doi: 10.1371/journal.ppat.1012616

Figure Lengend Snippet: (A-B) hSAECs were infected with RSV (MOI = 0.1) and viral titers were assessed 3 days post-infection in scenarios where OGG1 expression was either (A) down-regulated via siRNA or (B) completely eliminated by CRISPR/Cas9 knockout. NT, non-targeting siRNA. The knockout was overcompensated by ectopic expression of OGG1. The lower panel displays immunoblot analysis of OGG1 expression in cell lysates, with Actin serving as a loading control. (C) Following 1-hour post inoculation (MOI = 0.1), hSAECs were treated with TH5487, TH2840, or O8, and viral titers were determined by plaque assays (3 dpi). In D-I, hSAECs were infected with RSV (MOI = 0.5), and total RNA was extracted first at indicated times post-infection. Experiments include OGG1 knockout by CRISPR/Cas9 in hSAECs (D and G), down regulation of OGG1 expression by siRNA (E and H), and inhibition of OGG1’s reading function by TH5487 (F and I). (D-F) mRNA was isolated using Oligo dT beads, and G mRNA level was quantified by RT-qPCR. (G-I) After mRNA isolation, RSV gRNA levels were determined via RT-qPCR. n = 3. Statistical analysis was conducted using an unpaired Student’s t test, with results shown as means ± SD. Significance levels are indicated as * p <0.05, ** p < 0.01, *** p < 0.001. (J) Efficiency of primer extension on RNA template containing 8-oxoGua. Moloney murine leukemia virus reverse transcriptase (RT) was used in the assay. H 2 O 2 , 800 μM. OGG1, 50 nM. P indicates primer. E indicates extension.

Article Snippet: Specifically, GST-tagged OGG1 (TP720109, OriGene, Rockville, MD) was immobilized onto Glutathione Agarose, and subsequently incubated with varying concentrations of His-tagged nucleoprotein (NP-424V, Creative BioMart, Shirley, NY), or His-tagged M2-1 protein (purified locally).

Techniques: Infection, Expressing, CRISPR, Knock-Out, Western Blot, Control, Inhibition, Isolation, Quantitative RT-PCR, Virus, Reverse Transcription

Journal: PLOS Pathogens

Article Title: 8-Oxoguanine DNA Glycosylase1 conceals oxidized guanine in nucleoprotein-associated RNA of respiratory syncytial virus

doi: 10.1371/journal.ppat.1012616

Figure Lengend Snippet: Elevated ROS levels strategically alter host mechanisms to detect oxidative modifications in viral RNA. (A) OGG1 assumes a pivotal role in this process, co-opted by viral nucleoproteins to facilitate precise base pairing in progeny genomes. (B) Inhibiting OGG1’s ability to detect 8-oxoGua compromises the accuracy of viral replication, resulting in reduced viral progeny production. RdRp, RNA-dependent RNA polymerase. Figure created with Biorender.com .

Article Snippet: Specifically, GST-tagged OGG1 (TP720109, OriGene, Rockville, MD) was immobilized onto Glutathione Agarose, and subsequently incubated with varying concentrations of His-tagged nucleoprotein (NP-424V, Creative BioMart, Shirley, NY), or His-tagged M2-1 protein (purified locally).

Techniques:

Journal: iScience

Article Title: Small molecule inhibitor binds to NOD-like receptor family pyrin domain containing 3 and prevents inflammasome activation

doi: 10.1016/j.isci.2024.110459

Figure Lengend Snippet:

Article Snippet: Recombinant Human OGG1 protein , Abcam , ab98249.

Techniques: Virus, Recombinant, Enzyme-linked Immunosorbent Assay, SYBR Green Assay, Mutagenesis, Transfection, Plasmid Preparation, Knock-Out, Sonication, Software

Journal: Molecular cell

Article Title: Targeted and Persistent 8-Oxoguanine Base Damage at Telomeres Promotes Telomere Loss and Crisis

doi: 10.1016/j.molcel.2019.04.024

Figure Lengend Snippet: (A) Endogenous OGG1 and OGG1-EGFP (red) localization to telomeres (green) in HeLaFAP cells by anti-OGG1 IF and telo-FISH after dye + light.

Article Snippet: Human OGG1 , NovusBiologicals , Cal#NBP1–45318.

Techniques:

Journal: Molecular cell

Article Title: Targeted and Persistent 8-Oxoguanine Base Damage at Telomeres Promotes Telomere Loss and Crisis

doi: 10.1016/j.molcel.2019.04.024

Figure Lengend Snippet: (A) Immunoblot of OGG1 in parental HeLaFAP cells and two OGG1 KO clones. Actin was used as a loading control.

Article Snippet: Human OGG1 , NovusBiologicals , Cal#NBP1–45318.

Techniques: Western Blot, Clone Assay, Control

Journal: Molecular cell

Article Title: Targeted and Persistent 8-Oxoguanine Base Damage at Telomeres Promotes Telomere Loss and Crisis

doi: 10.1016/j.molcel.2019.04.024

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Human OGG1 , NovusBiologicals , Cal#NBP1–45318.

Techniques: Recombinant, Plasmid Preparation, Imaging, Clone Assay, Sequencing, shRNA, Software