recombinant human ogg1  (Novus Biologicals)

Journal: Journal of molecular biology

Article Title: Human CST Stimulates Base Excision Repair to Prevent the Accumulation of Oxidative DNA Damage

doi: 10.1016/j.jmb.2024.168672

Figure Lengend Snippet: (A) Representative images of proximity ligation assay (PLA) performed in HeLa cells with antibodies to STN1 or Pol β alone or in combination with and without H 2 O 2 treatment. Red, PLA foci; blue, DAPI. (B) Violin plot of PLA foci per nucleus. Results are representative of three independent, biological experiments. Bold dashed line represents the median, and dashed lines represent the first and third quartiles. (C) Proximity ligation assay (PLA) performed in HeLa cells with antibodies to STN1, OGG1, or in combination (STN1 + OGG1). n = 3 independent, biological experiments. Bold dashed line: median, dashed lines: first and third quartiles. (D) HEK293T cell extracts containing individually overexpressed CST components (either FLAG-CST, Myc-STN1 or FLAG-TEN1) that were either untreated or treated with 200 μM of H 2 O 2 were used to immunoprecipitate APE1, Pol β, FEN1 or LIGI and immunobloted with either anti-FLAG (for CTC1 and TEN1) or anti-Myc for STN1. Lysates were pre-treated with benzonase prior to coimmunoprecipitation. Proteins are identified in the Western blot analysis. (**** p < 0.0001).

Article Snippet: Recombinant human OGG1 was commercially purchased from Novus Biologicals (NBP1-45318).

Techniques: Proximity Ligation Assay, Western Blot

Journal: Journal of molecular biology

Article Title: Human CST Stimulates Base Excision Repair to Prevent the Accumulation of Oxidative DNA Damage

doi: 10.1016/j.jmb.2024.168672

Figure Lengend Snippet: (A) OGG1 (2 nM, lanes 2, 4–15; 50 nM, lane 3) lesion base removal and phosphodiester bone cleavage activity were measured on a random sequence DNA substrate (5 nM) containing an 8-oxo-guanine (8-oxo-G) residue in the presence of increasing concentrations of CTC1/STN1/TEN1 (250, 750, 1500 nM) or CST (5, 15, 60 nM) as denoted in the figure. The red asterisk shown on the substrate in the figure represents the location of the 32 P radiolabel and a G* represents an 8-oxo-G residue. Gel shown is representative of at least three independent experiments. (B) Graph of the fold increase in OGG1 cleavage activity for the lanes containing the highest concentration of the CST complex and individual subunits. The random substrate (U1:T1) was generated by annealing primers in a 1:2 ratio. (**** p < 0.0001).

Article Snippet: Recombinant human OGG1 was commercially purchased from Novus Biologicals (NBP1-45318).

Techniques: Activity Assay, Sequencing, Residue, Concentration Assay, Generated

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