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FIGURE 1. Stimulation of epithelial cells, RELA activation, DNA binding, and gene expression. (A) Time course of RELA activation. Detroit 562 cells were stimulated for 2 h with LPS, TNF-a, Pam2CSK4, or poly I:C, and RELA activation was determined with the NF-kB <t>p65</t> TF assay at 30-min interval for 2 h. Results are average luminescence readouts of two independent experiments. (B) Differential gene expression in response to different stimuli. After stimulation with LPS for 100 min, TNF-a for 70 min, Pam2CSK4 for 80 min, or poly I:C for 110 min, RNAs were isolated from Detroit 562 cells for RNA- seq analysis. Average Log2FC of biological duplicates were used for hierarchical clustering of genes DE in at least one condition. Sets of genes UP in poly I:C only or all conditions are highlighted. (C) Annotation of RELA binding sites. ChIP-seq experiment was performed in duplicates after treatment of Detroit 562 cells with LPS for 80 min, TNF-a for 50 min, Pam2CSK4 for 60 min, or poly I:C for 90 min. The number of peaks identiﬁed for each stimulus is reported on the right. Peaks were annotated; the bar chart shows the number of peaks in the different genomic features indicated. (D) UP, DOWN, or nonregulated (NO) genes after treatment with the four stimuli were assigned RELA ChIP-seq peaks located within 50 kb (Figure legend continues)

Antirela Ab, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 34 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/antirela+ab/Epo+siRNA/pm29959281-72-5-11
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antirela ab - by Bioz Stars, 2026-10

93/100 stars

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1) Product Images from "Variation in Genome-Wide NF-κB RELA Binding Sites upon Microbial Stimuli and Identification of a Virus Response Profile."

Article Title: Variation in Genome-Wide NF-κB RELA Binding Sites upon Microbial Stimuli and Identification of a Virus Response Profile.

Journal: Journal of immunology (Baltimore, Md. : 1950)

doi: 10.4049/jimmunol.1800246

Figure Legend Snippet: FIGURE 1. Stimulation of epithelial cells, RELA activation, DNA binding, and gene expression. (A) Time course of RELA activation. Detroit 562 cells were stimulated for 2 h with LPS, TNF-a, Pam2CSK4, or poly I:C, and RELA activation was determined with the NF-kB p65 TF assay at 30-min interval for 2 h. Results are average luminescence readouts of two independent experiments. (B) Differential gene expression in response to different stimuli. After stimulation with LPS for 100 min, TNF-a for 70 min, Pam2CSK4 for 80 min, or poly I:C for 110 min, RNAs were isolated from Detroit 562 cells for RNA- seq analysis. Average Log2FC of biological duplicates were used for hierarchical clustering of genes DE in at least one condition. Sets of genes UP in poly I:C only or all conditions are highlighted. (C) Annotation of RELA binding sites. ChIP-seq experiment was performed in duplicates after treatment of Detroit 562 cells with LPS for 80 min, TNF-a for 50 min, Pam2CSK4 for 60 min, or poly I:C for 90 min. The number of peaks identiﬁed for each stimulus is reported on the right. Peaks were annotated; the bar chart shows the number of peaks in the different genomic features indicated. (D) UP, DOWN, or nonregulated (NO) genes after treatment with the four stimuli were assigned RELA ChIP-seq peaks located within 50 kb (Figure legend continues)

Techniques Used: Activation Assay, Binding Assay, Gene Expression, Transcription Factor Assay, Isolation, RNA Sequencing, ChIP-sequencing

FIGURE 2. Comparison of RELA binding sites across stimuli. (A) Comparison of RELA binding sites location. Genomic coordinates of RELA ChIP-seq peaks called upon each stimulus were compared and considered overlapping when the maximum distance between two peak centers was ,500 bp. The Venn diagram shows the number of peaks overlapping across the four conditions. (B) Differential binding analysis. The heatmap shows K-means clustering of the ChIP-seq signal (pooled biological duplicates) across the four stimulations. DB regions were separated into six groups with the number of peaks in each group shown on the right. (C) Annotation of DB and non-DB peaks. The percentage of peaks in each genomic feature for both sets of peaks is reported. Pearson p values from x2 test between the two sets are reported. ***p # 0.001, ****p # 0.0001.

Figure Legend Snippet: FIGURE 2. Comparison of RELA binding sites across stimuli. (A) Comparison of RELA binding sites location. Genomic coordinates of RELA ChIP-seq peaks called upon each stimulus were compared and considered overlapping when the maximum distance between two peak centers was ,500 bp. The Venn diagram shows the number of peaks overlapping across the four conditions. (B) Differential binding analysis. The heatmap shows K-means clustering of the ChIP-seq signal (pooled biological duplicates) across the four stimulations. DB regions were separated into six groups with the number of peaks in each group shown on the right. (C) Annotation of DB and non-DB peaks. The percentage of peaks in each genomic feature for both sets of peaks is reported. Pearson p values from x2 test between the two sets are reported. ***p # 0.001, ****p # 0.0001.

Techniques Used: Comparison, Binding Assay, ChIP-sequencing

$FIGURE 3. Poly I:C–increased RELA binding. (A) GO analysis of Group 5 peaks. Using Genomic Regions Enrichment of Annotation Tools, genomic regions from Group 5 peaks were associated to the single nearest gene and GO analysis was performed. The top ﬁve biological process terms are reported in this ﬁgure together with their p value. (B) Gene expression of genes associated with Group 5 peaks. DE genes in at least one condition were assigned DB RELA peaks located within 50 kb upstream to 50 kb downstream of the gene. The boxplot shows Log2FC in each condition (average across biological duplicates) of the genes assigned to Group 5 peaks. The p values from a paired Wilcoxon test against the gene expression under poly I:C stimulation are indicated. (C) Motif analysis of Group 5 peaks. Known motifs enrichment was investigated in the set of Group 5 peaks against all RELA peaks identiﬁed across stimuli. Log10 (p values) of signiﬁcantly enriched motifs are reported in the bar graph. (D) De novo motif analysis on Group 5 peaks. Top unknown motif enriched in Group 5 peaks together with the best match from JASPAR database are represented. (E) Overlap with IRF ChIP-seq data. Binding sites for IRFs were extracted from the ENCODE data and overlapped with the RELA peaks from Group 5 (yellow), DB peaks from the other groups (dark gray), or non-DB peaks (light gray). The fraction of RELA peaks overlapping IRF binding sites are reported; the Pearson p values from a x2 test against the results for Group 5 peaks are indicated. *p # 0.05, **p # 0.01, ***p # 0.001, ****p # 0.0001.$
Figure Legend Snippet: FIGURE 3. Poly I:C–increased RELA binding. (A) GO analysis of Group 5 peaks. Using Genomic Regions Enrichment of Annotation Tools, genomic regions from Group 5 peaks were associated to the single nearest gene and GO analysis was performed. The top ﬁve biological process terms are reported in this ﬁgure together with their p value. (B) Gene expression of genes associated with Group 5 peaks. DE genes in at least one condition were assigned DB RELA peaks located within 50 kb upstream to 50 kb downstream of the gene. The boxplot shows Log2FC in each condition (average across biological duplicates) of the genes assigned to Group 5 peaks. The p values from a paired Wilcoxon test against the gene expression under poly I:C stimulation are indicated. (C) Motif analysis of Group 5 peaks. Known motifs enrichment was investigated in the set of Group 5 peaks against all RELA peaks identiﬁed across stimuli. Log10 (p values) of signiﬁcantly enriched motifs are reported in the bar graph. (D) De novo motif analysis on Group 5 peaks. Top unknown motif enriched in Group 5 peaks together with the best match from JASPAR database are represented. (E) Overlap with IRF ChIP-seq data. Binding sites for IRFs were extracted from the ENCODE data and overlapped with the RELA peaks from Group 5 (yellow), DB peaks from the other groups (dark gray), or non-DB peaks (light gray). The fraction of RELA peaks overlapping IRF binding sites are reported; the Pearson p values from a x2 test against the results for Group 5 peaks are indicated. *p # 0.05, **p # 0.01, ***p # 0.001, ****p # 0.0001.

Techniques Used: Binding Assay, Gene Expression, ChIP-sequencing

FIGURE 4. OASL locus as an example of stimulus-speciﬁc RELA target. (A) Genome browser view of RELA ChIP-seq signal under different stimuli. A RELA binding site with higher signal under poly I:C stimulation is detected ∼20 kb upstream of the OASL gene. (B) Validation of the poly I:C–increased peak by ChIP qPCR. Primers were designed to amplify the region highlighted in (A) (yellow). The bar chart shows the enrichment of RELA binding at this location as percentage of input recovered. (C) OASL expression upon ﬁve stimuli. Expression was determined by RT-qPCR and expressed as fold changes in OASL expression after stimulation as compared with untreated cells. (D) Inhibition of RELA binding. Cells were pretreated with BAY 11-8072 or DMSO followed by poly I:C stimulation or no treatment control. Binding of RELA at the region highlighted in yellow in (A) was investigated by ChIP qPCR and shown as the average input percentage recovered from the immunoprecipitation. (E) NF-kB regulation of OASL. Cells were pretreated with BAY 11-8072 or DMSO followed by poly I:C stimulation or no treatment control, and OASL expression was measured by RT-qPCR and represented as fold change of expression over the control. Data in (B)–(E) represents the average of two independent experiments and SD as error bars.

Figure Legend Snippet: FIGURE 4. OASL locus as an example of stimulus-speciﬁc RELA target. (A) Genome browser view of RELA ChIP-seq signal under different stimuli. A RELA binding site with higher signal under poly I:C stimulation is detected ∼20 kb upstream of the OASL gene. (B) Validation of the poly I:C–increased peak by ChIP qPCR. Primers were designed to amplify the region highlighted in (A) (yellow). The bar chart shows the enrichment of RELA binding at this location as percentage of input recovered. (C) OASL expression upon ﬁve stimuli. Expression was determined by RT-qPCR and expressed as fold changes in OASL expression after stimulation as compared with untreated cells. (D) Inhibition of RELA binding. Cells were pretreated with BAY 11-8072 or DMSO followed by poly I:C stimulation or no treatment control. Binding of RELA at the region highlighted in yellow in (A) was investigated by ChIP qPCR and shown as the average input percentage recovered from the immunoprecipitation. (E) NF-kB regulation of OASL. Cells were pretreated with BAY 11-8072 or DMSO followed by poly I:C stimulation or no treatment control, and OASL expression was measured by RT-qPCR and represented as fold change of expression over the control. Data in (B)–(E) represents the average of two independent experiments and SD as error bars.

Techniques Used: ChIP-sequencing, Binding Assay, Biomarker Discovery, ChIP-qPCR, Expressing, Quantitative RT-PCR, Inhibition, Control, Immunoprecipitation

Incubation:

Article Title: Variation in Genome-Wide NF-κB RELA Binding Sites upon Microbial Stimuli and Identification of a Virus Response Profile.
Article Snippet: Chromatin shearing was performed using a sonicator (Bioruptor, Diagenode) on a high setting for 45 cycles of 30 s ON/45 s OFF, leading to a fragment range of 100–250 bp. .. Chromatin was then incubated with antiRELA Ab (NF-kB p65 [c-20] sc-372; Santa Cruz Biotechnology) bound to protein G magnetic beads (Dyanabeads protein G; Thermo Fisher Scientific) prepared by mixing 4 mg of Ab with 40 ml of beads in PBS plus 0.5% BSA for 4–6 h at room temperature. ..

Magnetic Beads:

Immunoprecipitation:

Article Title: Adenosine is a negative regulator of NF-kappaB and MAPK signaling in human intestinal epithelial cells.
Article Snippet: Previous studies suggest that adenosine possesses anti-inXammatory properties, however, the mechanisms by which adenosine aVects immune function remain unclear, particularly in the intestine.. In this study, we hypothesized that adenosine directly aVects pro-inXammatory gene expression in intestinal epithelial cells through modulation of NFB signaling.. HT-29 cells were treated with adenosine prior to incubation with various stimuli and pro-inXammatory gene expression and signal transduction analyzed.

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Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Variation in Genome-Wide NF-κB RELA Binding Sites upon Microbial Stimuli and Identification of a Virus Response Profile.

doi: 10.4049/jimmunol.1800246

Figure Lengend Snippet: FIGURE 1. Stimulation of epithelial cells, RELA activation, DNA binding, and gene expression. (A) Time course of RELA activation. Detroit 562 cells were stimulated for 2 h with LPS, TNF-a, Pam2CSK4, or poly I:C, and RELA activation was determined with the NF-kB p65 TF assay at 30-min interval for 2 h. Results are average luminescence readouts of two independent experiments. (B) Differential gene expression in response to different stimuli. After stimulation with LPS for 100 min, TNF-a for 70 min, Pam2CSK4 for 80 min, or poly I:C for 110 min, RNAs were isolated from Detroit 562 cells for RNA- seq analysis. Average Log2FC of biological duplicates were used for hierarchical clustering of genes DE in at least one condition. Sets of genes UP in poly I:C only or all conditions are highlighted. (C) Annotation of RELA binding sites. ChIP-seq experiment was performed in duplicates after treatment of Detroit 562 cells with LPS for 80 min, TNF-a for 50 min, Pam2CSK4 for 60 min, or poly I:C for 90 min. The number of peaks identiﬁed for each stimulus is reported on the right. Peaks were annotated; the bar chart shows the number of peaks in the different genomic features indicated. (D) UP, DOWN, or nonregulated (NO) genes after treatment with the four stimuli were assigned RELA ChIP-seq peaks located within 50 kb (Figure legend continues)

Article Snippet: Chromatin was then incubated with antiRELA Ab (NF-kB p65 [c-20] sc-372; Santa Cruz Biotechnology) bound to protein G magnetic beads (Dyanabeads protein G; Thermo Fisher Scientific) prepared by mixing 4 mg of Ab with 40 ml of beads in PBS plus 0.5% BSA for 4–6 h at room temperature.

Techniques: Activation Assay, Binding Assay, Gene Expression, Transcription Factor Assay, Isolation, RNA Sequencing, ChIP-sequencing

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Variation in Genome-Wide NF-κB RELA Binding Sites upon Microbial Stimuli and Identification of a Virus Response Profile.

doi: 10.4049/jimmunol.1800246

Figure Lengend Snippet: FIGURE 2. Comparison of RELA binding sites across stimuli. (A) Comparison of RELA binding sites location. Genomic coordinates of RELA ChIP-seq peaks called upon each stimulus were compared and considered overlapping when the maximum distance between two peak centers was ,500 bp. The Venn diagram shows the number of peaks overlapping across the four conditions. (B) Differential binding analysis. The heatmap shows K-means clustering of the ChIP-seq signal (pooled biological duplicates) across the four stimulations. DB regions were separated into six groups with the number of peaks in each group shown on the right. (C) Annotation of DB and non-DB peaks. The percentage of peaks in each genomic feature for both sets of peaks is reported. Pearson p values from x2 test between the two sets are reported. ***p # 0.001, ****p # 0.0001.

Techniques: Comparison, Binding Assay, ChIP-sequencing

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Variation in Genome-Wide NF-κB RELA Binding Sites upon Microbial Stimuli and Identification of a Virus Response Profile.

doi: 10.4049/jimmunol.1800246

Figure Lengend Snippet: FIGURE 3. Poly I:C–increased RELA binding. (A) GO analysis of Group 5 peaks. Using Genomic Regions Enrichment of Annotation Tools, genomic regions from Group 5 peaks were associated to the single nearest gene and GO analysis was performed. The top ﬁve biological process terms are reported in this ﬁgure together with their p value. (B) Gene expression of genes associated with Group 5 peaks. DE genes in at least one condition were assigned DB RELA peaks located within 50 kb upstream to 50 kb downstream of the gene. The boxplot shows Log2FC in each condition (average across biological duplicates) of the genes assigned to Group 5 peaks. The p values from a paired Wilcoxon test against the gene expression under poly I:C stimulation are indicated. (C) Motif analysis of Group 5 peaks. Known motifs enrichment was investigated in the set of Group 5 peaks against all RELA peaks identiﬁed across stimuli. Log10 (p values) of signiﬁcantly enriched motifs are reported in the bar graph. (D) De novo motif analysis on Group 5 peaks. Top unknown motif enriched in Group 5 peaks together with the best match from JASPAR database are represented. (E) Overlap with IRF ChIP-seq data. Binding sites for IRFs were extracted from the ENCODE data and overlapped with the RELA peaks from Group 5 (yellow), DB peaks from the other groups (dark gray), or non-DB peaks (light gray). The fraction of RELA peaks overlapping IRF binding sites are reported; the Pearson p values from a x2 test against the results for Group 5 peaks are indicated. *p # 0.05, **p # 0.01, ***p # 0.001, ****p # 0.0001.

Techniques: Binding Assay, Gene Expression, ChIP-sequencing

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Variation in Genome-Wide NF-κB RELA Binding Sites upon Microbial Stimuli and Identification of a Virus Response Profile.

doi: 10.4049/jimmunol.1800246

Figure Lengend Snippet: FIGURE 4. OASL locus as an example of stimulus-speciﬁc RELA target. (A) Genome browser view of RELA ChIP-seq signal under different stimuli. A RELA binding site with higher signal under poly I:C stimulation is detected ∼20 kb upstream of the OASL gene. (B) Validation of the poly I:C–increased peak by ChIP qPCR. Primers were designed to amplify the region highlighted in (A) (yellow). The bar chart shows the enrichment of RELA binding at this location as percentage of input recovered. (C) OASL expression upon ﬁve stimuli. Expression was determined by RT-qPCR and expressed as fold changes in OASL expression after stimulation as compared with untreated cells. (D) Inhibition of RELA binding. Cells were pretreated with BAY 11-8072 or DMSO followed by poly I:C stimulation or no treatment control. Binding of RELA at the region highlighted in yellow in (A) was investigated by ChIP qPCR and shown as the average input percentage recovered from the immunoprecipitation. (E) NF-kB regulation of OASL. Cells were pretreated with BAY 11-8072 or DMSO followed by poly I:C stimulation or no treatment control, and OASL expression was measured by RT-qPCR and represented as fold change of expression over the control. Data in (B)–(E) represents the average of two independent experiments and SD as error bars.

Techniques: ChIP-sequencing, Binding Assay, Biomarker Discovery, ChIP-qPCR, Expressing, Quantitative RT-PCR, Inhibition, Control, Immunoprecipitation

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