Review

mouse monoclonal anti rna polymerase ii ctd repeat (Santa Cruz Biotechnology)

Name: mouse monoclonal anti rna polymerase ii ctd repeat
Brand: Santa Cruz Biotechnology
Rating: 97 (1641 reviews)

Santa Cruz Biotechnology is a verified supplier

About

News

Press Release

Team

Advisors

Partners

Contact

Bioz Stars

Bioz vStars

Buy from Supplier

Structured Review

Santa Cruz Biotechnology mouse monoclonal anti rna polymerase ii ctd repeat
Mouse Monoclonal Anti Rna Polymerase Ii Ctd Repeat, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 97/100, based on 1641 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/mouse monoclonal anti rna polymerase ii ctd repeat/product/Santa Cruz Biotechnology
Average 97 stars, based on 1641 article reviews

mouse monoclonal anti rna polymerase ii ctd repeat - by Bioz Stars, 2026-03

97/100 stars

Images

Image Search Results

Endogenous mouse heparanase (mHPSE) restricts MLV infection. (A) Relative mHPSE RNA levels were determined by RT-PCR in mouse embryonic fibroblasts (MEFs) wild type (WT) and MEF Hpse−/− (KO). (B–C) WT and KO cells were infected with MLV (0.02 MOI) and at the indicated time points post-infection cells were harvested. (B) MLV env DNA was measured by qPCR and normalized to mouse Gapdh . (C) MLV p30 levels in virus-containing media were analyzed by immunoblotting for the indicated protein. Representative immunoblotting results of three independent experiments are shown. All data presented as mean ± standard error of the mean. Statistical analysis by (A) one-sample t test or (B) multiple t tests, N = 3, ns: not significant, ** P ≤ 0.01, and **** P ≤ 0.0001.

Journal: mBio

Article Title: Heparanase, a host gene that potently restricts retrovirus transcription

doi: 10.1128/mbio.03252-24

Figure Lengend Snippet: Endogenous mouse heparanase (mHPSE) restricts MLV infection. (A) Relative mHPSE RNA levels were determined by RT-PCR in mouse embryonic fibroblasts (MEFs) wild type (WT) and MEF Hpse−/− (KO). (B–C) WT and KO cells were infected with MLV (0.02 MOI) and at the indicated time points post-infection cells were harvested. (B) MLV env DNA was measured by qPCR and normalized to mouse Gapdh . (C) MLV p30 levels in virus-containing media were analyzed by immunoblotting for the indicated protein. Representative immunoblotting results of three independent experiments are shown. All data presented as mean ± standard error of the mean. Statistical analysis by (A) one-sample t test or (B) multiple t tests, N = 3, ns: not significant, ** P ≤ 0.01, and **** P ≤ 0.0001.

Article Snippet: Samples containing 10 μg of DNA were then incubated overnight at 4°C with either anti-RNA Pol II antibody (mouse anti-Rpb1 CTD, Cell Signaling, 2629S), mouse anti-SP1 antibody (Santa Cruz Biotechnology, sc-17824X), or mouse IgG (Invitrogen, 31878) in ChIP dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM pH 8 Tris–HCl, and 0.167 M NaCl).

Techniques: Infection, Reverse Transcription Polymerase Chain Reaction, Virus, Western Blot

$Mouse heparanase (mHPSE) restricts retrovirus transcription initiation. ( A ) mHPSE is primarily localized in the nucleus. 293T cells were transfected with plasmids encoding mHPSE or co-transfected with mHPSE and an MLV infectious clone, and then fractionated into whole cell lysate (WC), cytosolic fraction ( C ), or nuclear fraction ( N ). Samples were then analyzed by immunoblotting for the indicated proteins. Representative immunoblotting results of three independent experiments are shown. ( B ) mHPSE does not affect particle infectivity of progeny virions. Mouse embryonic fibroblasts (MEFs) wild type (WT) and MEF Hpse−/− (KO) were infected with MLV (0.1 MOI), and equal amounts of the subsequently produced viruses were used to infect Mus dunni cells. At 24 h post-infection, MLV DNA levels were determined by qPCR and normalized to GADPH . ( C ) mHPSE does not affect MLV integration. 293T-MCAT-1 cells were transfected with either mHPSE-expressing or empty vector (EV) plasmids, and then infected in either the presence or absence of 100 nM Raltegravir (RAL) with equal amounts of luciferase reporter pseudoviruses bearing the MLV envelope. Viral DNA was measured by qPCR and normalized to gapdh . ( D ) mHPSE reduces levels of all MLV viral transcripts. 293T cells were co-transfected with an MLV infectious clone and either EV or increasing amounts of mHPSE-expressing plasmid. After 48 h, MLV RNA transcripts were quantified by RT-qPCR, with primers targeting the indicated regions. ( E ) Endogenous mHPSE restricts MLV expression. MEF WT and MEF KO cells were infected with equal amounts of MLV envelope pseudotyped luciferase reporter virus, and, at 2 dpi, luciferase expression was measured by luminescence assay and normalized to pseudovirus integration levels. ( F–G ) mHPSE does not affect the stability of MLV RNA. 293T cells were co-transfected with plasmids encoding MLV and either mHPSE or EV. Cells were then treated with 20 µg /mL of actinomycin D, and MLV RNA levels were quantified by RT-qPCR at the indicated time points post-treatment. ( F ) Data presented as mean MLV RNA normalized to GAPDH . ( G ) To measure the rate of RNA decay, MLV RNA values from F were normalized to the 0 h time point. ( H ) mHPSE decreases the association of RNA Pol II with the MLV LTR. WT and Hpse KO MEFs were infected with MLV envelope pseudotyped luciferase reporter virus. DNA was subsequently precipitated with an antibody targeting RNA Pol II (αRP) or an isotype control (IgG), and enrichment of RNA Pol II on the MLV LTR was determined by qPCR, with primers targeting the MLV LTR and normalized to LTR DNA present in input samples. All data in graphs presented as mean ± standard error of the mean. Statistical analysis was performed by ( B,F ) two-way analysis of variance (ANOVA), Sidak’s multiple-comparisons test, ( C–D ) one-way ANOVA, Dunnett’s multiple-comparisons or ( E, G, H ) unpaired t test, ( B–D ) N = 3 and ( E–H ) N = 4, ns: not significant, * P ≤ 0.05, ** P ≤ 0.01 , *** P ≤ 0.001, and **** P ≤ 0.0001.$

Journal: mBio

Article Title: Heparanase, a host gene that potently restricts retrovirus transcription

doi: 10.1128/mbio.03252-24

Figure Lengend Snippet: Mouse heparanase (mHPSE) restricts retrovirus transcription initiation. ( A ) mHPSE is primarily localized in the nucleus. 293T cells were transfected with plasmids encoding mHPSE or co-transfected with mHPSE and an MLV infectious clone, and then fractionated into whole cell lysate (WC), cytosolic fraction ( C ), or nuclear fraction ( N ). Samples were then analyzed by immunoblotting for the indicated proteins. Representative immunoblotting results of three independent experiments are shown. ( B ) mHPSE does not affect particle infectivity of progeny virions. Mouse embryonic fibroblasts (MEFs) wild type (WT) and MEF Hpse−/− (KO) were infected with MLV (0.1 MOI), and equal amounts of the subsequently produced viruses were used to infect Mus dunni cells. At 24 h post-infection, MLV DNA levels were determined by qPCR and normalized to GADPH . ( C ) mHPSE does not affect MLV integration. 293T-MCAT-1 cells were transfected with either mHPSE-expressing or empty vector (EV) plasmids, and then infected in either the presence or absence of 100 nM Raltegravir (RAL) with equal amounts of luciferase reporter pseudoviruses bearing the MLV envelope. Viral DNA was measured by qPCR and normalized to gapdh . ( D ) mHPSE reduces levels of all MLV viral transcripts. 293T cells were co-transfected with an MLV infectious clone and either EV or increasing amounts of mHPSE-expressing plasmid. After 48 h, MLV RNA transcripts were quantified by RT-qPCR, with primers targeting the indicated regions. ( E ) Endogenous mHPSE restricts MLV expression. MEF WT and MEF KO cells were infected with equal amounts of MLV envelope pseudotyped luciferase reporter virus, and, at 2 dpi, luciferase expression was measured by luminescence assay and normalized to pseudovirus integration levels. ( F–G ) mHPSE does not affect the stability of MLV RNA. 293T cells were co-transfected with plasmids encoding MLV and either mHPSE or EV. Cells were then treated with 20 µg /mL of actinomycin D, and MLV RNA levels were quantified by RT-qPCR at the indicated time points post-treatment. ( F ) Data presented as mean MLV RNA normalized to GAPDH . ( G ) To measure the rate of RNA decay, MLV RNA values from F were normalized to the 0 h time point. ( H ) mHPSE decreases the association of RNA Pol II with the MLV LTR. WT and Hpse KO MEFs were infected with MLV envelope pseudotyped luciferase reporter virus. DNA was subsequently precipitated with an antibody targeting RNA Pol II (αRP) or an isotype control (IgG), and enrichment of RNA Pol II on the MLV LTR was determined by qPCR, with primers targeting the MLV LTR and normalized to LTR DNA present in input samples. All data in graphs presented as mean ± standard error of the mean. Statistical analysis was performed by ( B,F ) two-way analysis of variance (ANOVA), Sidak’s multiple-comparisons test, ( C–D ) one-way ANOVA, Dunnett’s multiple-comparisons or ( E, G, H ) unpaired t test, ( B–D ) N = 3 and ( E–H ) N = 4, ns: not significant, * P ≤ 0.05, ** P ≤ 0.01 , *** P ≤ 0.001, and **** P ≤ 0.0001.

Techniques: Transfection, Western Blot, Infection, Produced, Expressing, Plasmid Preparation, Luciferase, Quantitative RT-PCR, Virus, Luminescence Assay, Control

FIGURE 2 | AnHV-1 DNA replication was positively correlated with recruitment of RNA pol II to vRCs. (A) DEF cells were infected with AnHV-1 at 1 MOI for 24 hpi. Fixed cells were stained for ICP4 (Red) and RNA Pol II (Green) to visualise the formation of replication compartments. Images were captured at 40× magnification. DEF cells were infected with AnHV-1 10 MOI for 9/12 hpi and with or without PAA (200 ug/ml) in figure (B–E). (B) Quantification of morphological changes of viral replication foci. Each ICP4-positive cell was classified into one of three categories: No structure, small spots (pre-vRCs), or large spots (vRCs). (C) Images of replication factories are shown in each panel and arrows indicate magnified cell. (D, E) Manual cell counting was performed to quantify the morphological changes of vRCs induced by inhibiting vDNA replication. Under 40× magnifi- cation, three random fields were selected, and the number of ICP4-positive cells co-localised with RNA Pol II, along with associated morphological changes, was counted. (F) DEF cells were infected with AnHV-1 at 10 MOI and treated with or without PAA (200 ug/ml). Cells were harvested 9 hpi for CUT&Tag assay. Changes in RNA pol II occupancy at each period gene were assessed by qPCR. The data were analysed by the t-test. Each column was obtained from two independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns: Not significant.

Journal: Cell proliferation

Article Title: The S-Phase Arrest of Host Cells Caused by an Alpha-Herpesvirus Genome Replication Facilitates Viral Recruitment of RNA Polymerase II to Transcribe Viral Genes.

doi: 10.1111/cpr.13811

Figure Lengend Snippet: FIGURE 2 | AnHV-1 DNA replication was positively correlated with recruitment of RNA pol II to vRCs. (A) DEF cells were infected with AnHV-1 at 1 MOI for 24 hpi. Fixed cells were stained for ICP4 (Red) and RNA Pol II (Green) to visualise the formation of replication compartments. Images were captured at 40× magnification. DEF cells were infected with AnHV-1 10 MOI for 9/12 hpi and with or without PAA (200 ug/ml) in figure (B–E). (B) Quantification of morphological changes of viral replication foci. Each ICP4-positive cell was classified into one of three categories: No structure, small spots (pre-vRCs), or large spots (vRCs). (C) Images of replication factories are shown in each panel and arrows indicate magnified cell. (D, E) Manual cell counting was performed to quantify the morphological changes of vRCs induced by inhibiting vDNA replication. Under 40× magnifi- cation, three random fields were selected, and the number of ICP4-positive cells co-localised with RNA Pol II, along with associated morphological changes, was counted. (F) DEF cells were infected with AnHV-1 at 10 MOI and treated with or without PAA (200 ug/ml). Cells were harvested 9 hpi for CUT&Tag assay. Changes in RNA pol II occupancy at each period gene were assessed by qPCR. The data were analysed by the t-test. Each column was obtained from two independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns: Not significant.

Article Snippet: The primary antibody used was anti- RNA pol II CTD (2629, CST), and the secondary antibody was the Unconjugated Secondary Antibody nloaded from https://onlinelibrary.w iley.com /doi/10.1111/cpr.13811 by IN A SP - N E PA L , W iley O nline L ibrary on [02/02/2025].

Techniques: Infection, Staining, Cell Counting

FIGURE 3 | Suppression of AnHV-1 genome replication extentsively decreased viral mRNA levels. (A) DEF cells were infected with AnHV-1 at 1 MOI, with or without PAA (200 μg/mL) treatment. Samples were collected at 12 hpi for RNA-Seq. Normalised read counts along the AnHV-1 ge- nome were displayed using the Integrative Genomics Viewer (IGV). The data shown in Figure 3A represent one representative replicate from each experimental group. Blue indicates the AnHV-1 infection group, while red represents the group treated with PAA. (B) Distribution of RNA Pol II in duck and AnHV-1 genomes in AnHV-1 infection with PAA or without. (C) Determination the mRNA level of representvie genes at 12/24/48/72 hpi after with or without PAA treatment by RT-qPCR. Each sample was repeated three times and the normalised gene is 18sRNA. (D) Changes in protein expression levels of ICP8 and gI were measured following drug treatment, normalised by GAPDH as an internal reference gene treatment. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns: Not significant.

Journal: Cell proliferation

Article Title: The S-Phase Arrest of Host Cells Caused by an Alpha-Herpesvirus Genome Replication Facilitates Viral Recruitment of RNA Polymerase II to Transcribe Viral Genes.

doi: 10.1111/cpr.13811

Figure Lengend Snippet: FIGURE 3 | Suppression of AnHV-1 genome replication extentsively decreased viral mRNA levels. (A) DEF cells were infected with AnHV-1 at 1 MOI, with or without PAA (200 μg/mL) treatment. Samples were collected at 12 hpi for RNA-Seq. Normalised read counts along the AnHV-1 ge- nome were displayed using the Integrative Genomics Viewer (IGV). The data shown in Figure 3A represent one representative replicate from each experimental group. Blue indicates the AnHV-1 infection group, while red represents the group treated with PAA. (B) Distribution of RNA Pol II in duck and AnHV-1 genomes in AnHV-1 infection with PAA or without. (C) Determination the mRNA level of representvie genes at 12/24/48/72 hpi after with or without PAA treatment by RT-qPCR. Each sample was repeated three times and the normalised gene is 18sRNA. (D) Changes in protein expression levels of ICP8 and gI were measured following drug treatment, normalised by GAPDH as an internal reference gene treatment. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns: Not significant.

Techniques: Infection, RNA Sequencing, Quantitative RT-PCR, Expressing

FIGURE 6 | Arrest at the S phase of the cell cycle to promote AnHV-1 hijacking RNA pol II. (A) DEF cells were inoculated with AnHV-1 at 0.01 MOI, with or without PAA treatment (200 μg/mL). At 24 hpi, fixed cells were stained for ICP8 (red) and RNA pol II (green). Nuculei were classified as structureless, small foci, or large foci. (B) Images of morphological changes of viral replication foci are shown in each panel. The fields of view were captured at 40× magnification. (C) Manual cell counting method to quantify the number of co-localization of ICP8 and RNA pol II and morphologi- cal changes in vRCs. (D) DEF cells were treated with 1 mM thymidine or no drug for 24h, and then infected with 0.01 MOI AnHV-1. At 24 hpi, cells were harvested for CUT&Tag assay. Changes in RNA pol II occupancy at each period genes were detected by qPCR. The data were analysed by the t-test. Each column was obtained from two independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns: Not significant.

Journal: Cell proliferation

Article Title: The S-Phase Arrest of Host Cells Caused by an Alpha-Herpesvirus Genome Replication Facilitates Viral Recruitment of RNA Polymerase II to Transcribe Viral Genes.

doi: 10.1111/cpr.13811

Figure Lengend Snippet: FIGURE 6 | Arrest at the S phase of the cell cycle to promote AnHV-1 hijacking RNA pol II. (A) DEF cells were inoculated with AnHV-1 at 0.01 MOI, with or without PAA treatment (200 μg/mL). At 24 hpi, fixed cells were stained for ICP8 (red) and RNA pol II (green). Nuculei were classified as structureless, small foci, or large foci. (B) Images of morphological changes of viral replication foci are shown in each panel. The fields of view were captured at 40× magnification. (C) Manual cell counting method to quantify the number of co-localization of ICP8 and RNA pol II and morphologi- cal changes in vRCs. (D) DEF cells were treated with 1 mM thymidine or no drug for 24h, and then infected with 0.01 MOI AnHV-1. At 24 hpi, cells were harvested for CUT&Tag assay. Changes in RNA pol II occupancy at each period genes were detected by qPCR. The data were analysed by the t-test. Each column was obtained from two independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns: Not significant.

Techniques: Staining, Cell Counting, Infection

A–B) ChIP-qPCR showing the enrichment of RNA polymerase II (RNA pol II) at the AalERV1 LTR (A) and the enrichment of H3K9me3 in the transposon body (B). Fold changes were calculated as the enrichment in ChIP over the respective input sample and internally normalized over a gene desert region (NW_021837055.1_88,280-89,336). Data represent the mean ± SD of two biological replicates in two independent ChIP experiments. C) Coverage of H3K9me3 enrichment over input of the ChIP-seq reads mapping to the combined Ae. albopictus repeat annotation consisting of Ae. albopictus repeat annotation and the AaERV1 consensus sequence. Log 2 enrichment of the H3K9me3 signal is shown across all repeat elements annotated in the Ae. albopictus repeat annotation (left), LTR Gypsy-family 3394 (middle), and the AaERV1 consensus sequence (right). Coverage was calculated over 100 bp windows with smoothening over 300 bp windows, and enrichment was calculated by taking the log 2 of H3K9me3 signal over input signal. ChIP-seq and input signals from individual samples are shown in Supplementary Figure S7C. See also Figure S5 and S7.

Journal: bioRxiv

Article Title: The piRNA pathway mediates transcriptional silencing of LTR retrotransposons in the soma and germline of Aedes mosquitoes

doi: 10.1101/2024.11.28.623418

Figure Lengend Snippet: A–B) ChIP-qPCR showing the enrichment of RNA polymerase II (RNA pol II) at the AalERV1 LTR (A) and the enrichment of H3K9me3 in the transposon body (B). Fold changes were calculated as the enrichment in ChIP over the respective input sample and internally normalized over a gene desert region (NW_021837055.1_88,280-89,336). Data represent the mean ± SD of two biological replicates in two independent ChIP experiments. C) Coverage of H3K9me3 enrichment over input of the ChIP-seq reads mapping to the combined Ae. albopictus repeat annotation consisting of Ae. albopictus repeat annotation and the AaERV1 consensus sequence. Log 2 enrichment of the H3K9me3 signal is shown across all repeat elements annotated in the Ae. albopictus repeat annotation (left), LTR Gypsy-family 3394 (middle), and the AaERV1 consensus sequence (right). Coverage was calculated over 100 bp windows with smoothening over 300 bp windows, and enrichment was calculated by taking the log 2 of H3K9me3 signal over input signal. ChIP-seq and input signals from individual samples are shown in Supplementary Figure S7C. See also Figure S5 and S7.

Article Snippet: Commercially available antibodies were used for immunoprecipitation: H3K9me3 polyclonal ab (C15410193, Diagenode) and RNA pol II 8WG16 mouse monoclonal ab (sc-56767, Santa Cruz).

Techniques: ChIP-qPCR, ChIP-sequencing, Sequencing

a Schematic representation of the workflow for the identification of RAD52 interacting proteins. HA-control and HA-RAD52 immunoprecipitation was performed in HEK293T cells using α-HA tagged magnetic beads for the pulldown followed by Mass spectrometry (MS). b Volcano plot of the proteins identified in RAD52 IP-MS in n = 3 biologically independent experiments. Mean log2 fold change in protein intensities on the x-axis of all replicates between HA and HA-RAD52 are plotted against the −log10 adjusted p -value (Student’s two-sided t -test with equal variance) on the y -axis. 212 proteins were identified to be significantly enriched. Significantly enriched proteins in blue ( p < 0.05) and non-significant in grey. c Co-immunoprecipitation of endogenous RAD52 binding proteins in HeLa cells. RAD52 and IgG antibodies were used to immuno-precipitate proteins and analyzed by immunoblotting with indicated antibodies. Results reproducible for at least 2 biological replicates. d Schematic representation of PLA to visualize proximity of RAD52 protein and RNA Pol II. e Representative images of the nuclear PLA foci (α-RAD52: α-RNA Pol II S2) across stated conditions (Scale bar 10 µM). f Quantitative analysis of nuclear PLA foci from ( e ) Data are plotted as mean ± SEM. The data presented shows ≥ 500 nuclei from 3 biological replicates; p -values calculated using unpaired two tailed t -tests. g Metagene plots showing the distribution of the RNA Pol II and RAD52 Chromatin immunoprecipitation sequencing (ChIP-seq) peaks (IP/input) in HeLa cells across genes and the flanking regions ( ± 10 kb). TSS: Transcription Start Site, TES: Transcription End Site. h Heatmap representing RNA Pol II and RAD52 ChIP-seq tracks, centered at the TSS and TES ± 10 kb, and rank-ordered according to RNA Pol II occupancy. i Bar chart showing how RNA Pol II and RAD52 peaks are distributed across different genomic regions as indicated. Peaks were obtained with MACS2. Genome wide distribution is shown on top for comparison. j Venn diagram showing the overlap of peaks RNA Pol II ChIP and RAD52 ChIP according to MACS2 across the genome. k A representative snapshot of chromosome 19 depicting RNA Pol II (red) and RAD52 (green) ChIP binding sites in control HeLa cells. Input DNA (grey) represents a negative control for background normalization. Schematics in Fig. 1 ( a ) and ( d ) were created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license. Source data are provided as a Source Data file.

Journal: Nature Communications

Article Title: RAD52 resolves transcription-replication conflicts to mitigate R-loop induced genome instability

doi: 10.1038/s41467-024-51784-x

Figure Lengend Snippet: a Schematic representation of the workflow for the identification of RAD52 interacting proteins. HA-control and HA-RAD52 immunoprecipitation was performed in HEK293T cells using α-HA tagged magnetic beads for the pulldown followed by Mass spectrometry (MS). b Volcano plot of the proteins identified in RAD52 IP-MS in n = 3 biologically independent experiments. Mean log2 fold change in protein intensities on the x-axis of all replicates between HA and HA-RAD52 are plotted against the −log10 adjusted p -value (Student’s two-sided t -test with equal variance) on the y -axis. 212 proteins were identified to be significantly enriched. Significantly enriched proteins in blue ( p < 0.05) and non-significant in grey. c Co-immunoprecipitation of endogenous RAD52 binding proteins in HeLa cells. RAD52 and IgG antibodies were used to immuno-precipitate proteins and analyzed by immunoblotting with indicated antibodies. Results reproducible for at least 2 biological replicates. d Schematic representation of PLA to visualize proximity of RAD52 protein and RNA Pol II. e Representative images of the nuclear PLA foci (α-RAD52: α-RNA Pol II S2) across stated conditions (Scale bar 10 µM). f Quantitative analysis of nuclear PLA foci from ( e ) Data are plotted as mean ± SEM. The data presented shows ≥ 500 nuclei from 3 biological replicates; p -values calculated using unpaired two tailed t -tests. g Metagene plots showing the distribution of the RNA Pol II and RAD52 Chromatin immunoprecipitation sequencing (ChIP-seq) peaks (IP/input) in HeLa cells across genes and the flanking regions ( ± 10 kb). TSS: Transcription Start Site, TES: Transcription End Site. h Heatmap representing RNA Pol II and RAD52 ChIP-seq tracks, centered at the TSS and TES ± 10 kb, and rank-ordered according to RNA Pol II occupancy. i Bar chart showing how RNA Pol II and RAD52 peaks are distributed across different genomic regions as indicated. Peaks were obtained with MACS2. Genome wide distribution is shown on top for comparison. j Venn diagram showing the overlap of peaks RNA Pol II ChIP and RAD52 ChIP according to MACS2 across the genome. k A representative snapshot of chromosome 19 depicting RNA Pol II (red) and RAD52 (green) ChIP binding sites in control HeLa cells. Input DNA (grey) represents a negative control for background normalization. Schematics in Fig. 1 ( a ) and ( d ) were created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license. Source data are provided as a Source Data file.

Article Snippet: Antibodies used in ChIP assays included: α-RNA Pol II (Cell Signaling 2629S), α-γH2AX (Abcam, #ab2893), α-RAD52 (Santa Cruz Biotechnology, #sc-365341), or α-S9.6 (Kerafast, #ENH001) and normal mouse IgG (Millipore-Sigma, #12-371).

Techniques: Control, Immunoprecipitation, Magnetic Beads, Mass Spectrometry, Protein-Protein interactions, Binding Assay, Western Blot, Two Tailed Test, ChIP-sequencing, Genome Wide, Comparison, Negative Control

a Representative snapshot of chromosome 9 depicting RNA Pol II occupancy from ChIP-seq analysis (IP/input) in siNT (red) and siRAD52 (dark red) transfected HeLa cells. ( b ) Metagene plot showing the distribution of the RNA Pol II occupancy at the TSS and flanking regions ( ± 10 kb) of genes with overlapping RNA Pol II and RAD52 peaks. Plots shown: siNT (control) and siRAD52 transfected HeLa cells. ( c ) ChIP-seq of RNA Pol II (red), RAD52 (green) and S9.6 (R-loops; blue) occupancy in control HeLa cells. Representative snapshot of chromosomes 21 are shown. Input (grey) DNA as negative control for background normalization. d Venn diagram of the percentage of genes overlapping with RNA Pol II, RAD52 and S9.6 ChIP peaks (MACS2). e Representative images of S9.6 immunostaining to detect R-loops in siNT (control) and siRAD52 transfected HeLa cells. RNase H treatment was added as a negative control to eliminate R-loops (Scale bar 10 µM). f Quantitative analysis of nuclear S9.6 foci across stated conditions from ( e ). Data plotted as box and whiskers. Boxes extend from the 25th–75th percentiles, with the median displayed as a line. The whiskers mark the minimum (1 percentile) and maximum (99 th percentile). The data presented shows ≥ 500 nuclei from 3 biological replicates; p -values calculated using unpaired two tailed t -tests. g Schematic representation of PLA to visualize proximity of PCNA and RNA Pol II to measure TRCs. The schematic illustration was created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license. h Representative images of the nuclear PLA foci (α-PCNA: α-RNA Pol II S2) across stated conditions (Scale bar 10 µM). i Quantitative analysis of nuclear PLA foci from ( h ). Data are plotted as mean ± SEM. The data presented shows ≥ 500 nuclei from 3 biological replicates; p -values calculated using unpaired two tailed t -tests. Source data are provided as a Source Data file.

Journal: Nature Communications

Article Title: RAD52 resolves transcription-replication conflicts to mitigate R-loop induced genome instability

doi: 10.1038/s41467-024-51784-x

Figure Lengend Snippet: a Representative snapshot of chromosome 9 depicting RNA Pol II occupancy from ChIP-seq analysis (IP/input) in siNT (red) and siRAD52 (dark red) transfected HeLa cells. ( b ) Metagene plot showing the distribution of the RNA Pol II occupancy at the TSS and flanking regions ( ± 10 kb) of genes with overlapping RNA Pol II and RAD52 peaks. Plots shown: siNT (control) and siRAD52 transfected HeLa cells. ( c ) ChIP-seq of RNA Pol II (red), RAD52 (green) and S9.6 (R-loops; blue) occupancy in control HeLa cells. Representative snapshot of chromosomes 21 are shown. Input (grey) DNA as negative control for background normalization. d Venn diagram of the percentage of genes overlapping with RNA Pol II, RAD52 and S9.6 ChIP peaks (MACS2). e Representative images of S9.6 immunostaining to detect R-loops in siNT (control) and siRAD52 transfected HeLa cells. RNase H treatment was added as a negative control to eliminate R-loops (Scale bar 10 µM). f Quantitative analysis of nuclear S9.6 foci across stated conditions from ( e ). Data plotted as box and whiskers. Boxes extend from the 25th–75th percentiles, with the median displayed as a line. The whiskers mark the minimum (1 percentile) and maximum (99 th percentile). The data presented shows ≥ 500 nuclei from 3 biological replicates; p -values calculated using unpaired two tailed t -tests. g Schematic representation of PLA to visualize proximity of PCNA and RNA Pol II to measure TRCs. The schematic illustration was created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license. h Representative images of the nuclear PLA foci (α-PCNA: α-RNA Pol II S2) across stated conditions (Scale bar 10 µM). i Quantitative analysis of nuclear PLA foci from ( h ). Data are plotted as mean ± SEM. The data presented shows ≥ 500 nuclei from 3 biological replicates; p -values calculated using unpaired two tailed t -tests. Source data are provided as a Source Data file.

Techniques: ChIP-sequencing, Transfection, Control, Negative Control, Immunostaining, Two Tailed Test

a Schematics of the domain structures of wild type (WT) - RAD52 protein and C-terminal (ΔC) deleted RAD52 (Δ302-410 amino acids). From N-terminal to C-terminal, RAD52 protein has DNA binding domain, RPA binding domain, RAD52 binding domain, RNA Pol II binding domain and a nuclear localization signal (NLS). The domains are not drawn to scale. b Western blot confirming the expression of HA-RAD52 WT and HA-RAD52 ΔC . Results reproducible for at least 2 biological replicates. c (Left) Scheme of the single stranded annealing (SSA) reporter system: The SSA-GFP reporter contains a 5′ fragment of the GFP (5′-GFP) gene, and a 3′ fragment of the GFP (3′-GFP) with an I-SceI site. Repair of the I-SceI-induced DSB by SSA leads to formation of GFP+ cells. (Middle) Quantification of SSA repair assay in WT and RAD52 −/− HCT116 cells. (Right) Quantification of SSA repair assay in RAD52 −/− HCT116 cells with overexpression of either RAD52 WT or RAD52 ΔC (n = 4 biological replicates). d (Left) Scheme of the homology dependent recombination (HDR) reporter system The HDR-GFP reporter system contains the GFP gene interrupted by a I-SceI site, and a fragment of the GFP with truncated 3′- and 5′-terminus. Repair of the I-SceI-induced DSB by HDR leads to formation of GFP+ cells. (Middle) Quantification of HDR repair assay in WT and RAD52 −/− HCT116 cells. (Right) Quantification of HDR repair assay in RAD52 −/− HCT116 cells with overexpression of either RAD52 WT or RAD52 ΔC . (n = 5 biological replicates). e Schematic representation of PLA to visualize proximity of HA-tagged RAD52 (HA-RAD52) and RNA Pol II. f Representative images of the nuclear PLA foci (α-HA: α-RNA Pol II S2) across stated conditions with overexpression of either RAD52 WT or RAD52 ΔC (Scale bar 10 µM). g Quantitative analysis of nuclear PLA foci across stated conditions described in ( f ). The data presented shows ≥ 500 nuclei from 3 biological replicates. h Schematic representation of PLA to visualize proximity of PCNA and RNA Pol II to measure TRCs. i Representative images of the nuclear PLA foci (α-PCNA: α-RNA Pol II S2) across stated conditions with overexpression of either RAD52 WT or RAD52 ΔC in HeLa cells (Scale bar 10 µM). j Quantitative analysis of nuclear PLA foci from across stated conditions described in ( i ). The data presented shows ≥ 500 nuclei from 3 biological replicates. In Fig. 3 ( c ) ( d ) ( g ) and ( j ), data are plotted as mean ± SEM and p -values calculated using unpaired two tailed t -tests. Schematics in Fig. 3 ( a ) ( c ) ( d ) ( e ) and ( h ) were created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license. Source data are provided as a Source Data file.

Journal: Nature Communications

Article Title: RAD52 resolves transcription-replication conflicts to mitigate R-loop induced genome instability

doi: 10.1038/s41467-024-51784-x

Figure Lengend Snippet: a Schematics of the domain structures of wild type (WT) - RAD52 protein and C-terminal (ΔC) deleted RAD52 (Δ302-410 amino acids). From N-terminal to C-terminal, RAD52 protein has DNA binding domain, RPA binding domain, RAD52 binding domain, RNA Pol II binding domain and a nuclear localization signal (NLS). The domains are not drawn to scale. b Western blot confirming the expression of HA-RAD52 WT and HA-RAD52 ΔC . Results reproducible for at least 2 biological replicates. c (Left) Scheme of the single stranded annealing (SSA) reporter system: The SSA-GFP reporter contains a 5′ fragment of the GFP (5′-GFP) gene, and a 3′ fragment of the GFP (3′-GFP) with an I-SceI site. Repair of the I-SceI-induced DSB by SSA leads to formation of GFP+ cells. (Middle) Quantification of SSA repair assay in WT and RAD52 −/− HCT116 cells. (Right) Quantification of SSA repair assay in RAD52 −/− HCT116 cells with overexpression of either RAD52 WT or RAD52 ΔC (n = 4 biological replicates). d (Left) Scheme of the homology dependent recombination (HDR) reporter system The HDR-GFP reporter system contains the GFP gene interrupted by a I-SceI site, and a fragment of the GFP with truncated 3′- and 5′-terminus. Repair of the I-SceI-induced DSB by HDR leads to formation of GFP+ cells. (Middle) Quantification of HDR repair assay in WT and RAD52 −/− HCT116 cells. (Right) Quantification of HDR repair assay in RAD52 −/− HCT116 cells with overexpression of either RAD52 WT or RAD52 ΔC . (n = 5 biological replicates). e Schematic representation of PLA to visualize proximity of HA-tagged RAD52 (HA-RAD52) and RNA Pol II. f Representative images of the nuclear PLA foci (α-HA: α-RNA Pol II S2) across stated conditions with overexpression of either RAD52 WT or RAD52 ΔC (Scale bar 10 µM). g Quantitative analysis of nuclear PLA foci across stated conditions described in ( f ). The data presented shows ≥ 500 nuclei from 3 biological replicates. h Schematic representation of PLA to visualize proximity of PCNA and RNA Pol II to measure TRCs. i Representative images of the nuclear PLA foci (α-PCNA: α-RNA Pol II S2) across stated conditions with overexpression of either RAD52 WT or RAD52 ΔC in HeLa cells (Scale bar 10 µM). j Quantitative analysis of nuclear PLA foci from across stated conditions described in ( i ). The data presented shows ≥ 500 nuclei from 3 biological replicates. In Fig. 3 ( c ) ( d ) ( g ) and ( j ), data are plotted as mean ± SEM and p -values calculated using unpaired two tailed t -tests. Schematics in Fig. 3 ( a ) ( c ) ( d ) ( e ) and ( h ) were created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license. Source data are provided as a Source Data file.

Techniques: Binding Assay, Western Blot, Expressing, Over Expression, Two Tailed Test

a Schematic representation of PLA to visualize proximity of RAD52 and TOP2A. b Representative images of the nuclear PLA foci (α-RAD52: α-TOP2A) in siNT (control) and siAQR transfected HeLa cells (Scale bar 10 µM). c Quantitative analysis of nuclear PLA foci across stated conditions described in ( b ). The data presented shows ≥ 500 nuclei from 3 biological replicates. d Schematic representation of PLA to visualize proximity of HA-tagged RAD52 (HA-RAD52) and TOP2A. e Representative images of the nuclear PLA foci (α-HA: α-TOP2A) in siRAD52 (5’UTR) transfected HeLa cells with overexpression of either RAD52 WT or RAD52 ΔC (Scale bar 10 µM). f Quantitative analysis of nuclear PLA foci across stated conditions described in ( e ). The data presented shows ≥ 500 nuclei from 3 biological replicates. g Representative images of S9.6 immunostaining to detect R-loops in siNT (control) and siTOP2A transfected HeLa cells. RNase H treatment was added as a negative control to eliminate R-loops (Scale bar 10 µM). h Quantitative analysis of nuclear S9.6 foci across stated conditions from ( g ). Data plotted as box and whiskers. Boxes extend from the 25th to 75th percentiles, with the median displayed as a line. The whiskers mark the minimum (1 percentile) and maximum (99th percentile). The data presented shows ≥ 500 nuclei from 3 biological replicates; p -values calculated using unpaired two tailed t -tests. i Schematic representation of PLA to visualize proximity of PCNA and RNA Pol II to measure TRCs. j Representative images of the nuclear PLA foci (PCNA: RNA Pol II S2) in siNT (control) and siTOP2A transfected HeLa cells (Scale bar 10 µM). k Quantitative analysis of nuclear PLA foci across stated conditions described in ( j ). The data presented shows ≥ 500 nuclei from 3 biological replicates ( l ) Schematic representation of PLA to visualize proximity of S9.6 and TOP2A. m Representative images of the nuclear PLA foci (α-S9.6: α-TOP2A) in siNT (control), siRAD52 and siAQR transfected HeLa cells (Scale bar 10 µM). n Quantitative analysis of nuclear PLA foci across stated conditions described in (m) normalized to siNT. The data presented shows ≥ 500 nuclei from 3 biological replicates. o Mechanistic model of RAD52 role in preventing transcription-replication conflicts. In Fig. 4 ( c – k ) and ( n ), data are plotted as mean ± SEM and p -values calculated using unpaired two tailed t -tests. Schematics in Fig. 4 ( a ) ( d ) ( i ) ( l ) and ( o ) were created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license. Source data are provided as a Source Data file.

Journal: Nature Communications

Article Title: RAD52 resolves transcription-replication conflicts to mitigate R-loop induced genome instability

doi: 10.1038/s41467-024-51784-x

Figure Lengend Snippet: a Schematic representation of PLA to visualize proximity of RAD52 and TOP2A. b Representative images of the nuclear PLA foci (α-RAD52: α-TOP2A) in siNT (control) and siAQR transfected HeLa cells (Scale bar 10 µM). c Quantitative analysis of nuclear PLA foci across stated conditions described in ( b ). The data presented shows ≥ 500 nuclei from 3 biological replicates. d Schematic representation of PLA to visualize proximity of HA-tagged RAD52 (HA-RAD52) and TOP2A. e Representative images of the nuclear PLA foci (α-HA: α-TOP2A) in siRAD52 (5’UTR) transfected HeLa cells with overexpression of either RAD52 WT or RAD52 ΔC (Scale bar 10 µM). f Quantitative analysis of nuclear PLA foci across stated conditions described in ( e ). The data presented shows ≥ 500 nuclei from 3 biological replicates. g Representative images of S9.6 immunostaining to detect R-loops in siNT (control) and siTOP2A transfected HeLa cells. RNase H treatment was added as a negative control to eliminate R-loops (Scale bar 10 µM). h Quantitative analysis of nuclear S9.6 foci across stated conditions from ( g ). Data plotted as box and whiskers. Boxes extend from the 25th to 75th percentiles, with the median displayed as a line. The whiskers mark the minimum (1 percentile) and maximum (99th percentile). The data presented shows ≥ 500 nuclei from 3 biological replicates; p -values calculated using unpaired two tailed t -tests. i Schematic representation of PLA to visualize proximity of PCNA and RNA Pol II to measure TRCs. j Representative images of the nuclear PLA foci (PCNA: RNA Pol II S2) in siNT (control) and siTOP2A transfected HeLa cells (Scale bar 10 µM). k Quantitative analysis of nuclear PLA foci across stated conditions described in ( j ). The data presented shows ≥ 500 nuclei from 3 biological replicates ( l ) Schematic representation of PLA to visualize proximity of S9.6 and TOP2A. m Representative images of the nuclear PLA foci (α-S9.6: α-TOP2A) in siNT (control), siRAD52 and siAQR transfected HeLa cells (Scale bar 10 µM). n Quantitative analysis of nuclear PLA foci across stated conditions described in (m) normalized to siNT. The data presented shows ≥ 500 nuclei from 3 biological replicates. o Mechanistic model of RAD52 role in preventing transcription-replication conflicts. In Fig. 4 ( c – k ) and ( n ), data are plotted as mean ± SEM and p -values calculated using unpaired two tailed t -tests. Schematics in Fig. 4 ( a ) ( d ) ( i ) ( l ) and ( o ) were created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license. Source data are provided as a Source Data file.

Techniques: Control, Transfection, Over Expression, Immunostaining, Negative Control, Two Tailed Test

Review

Structured Review

Images

Similar Products

Image Search Results