Journal: Molecular and Cellular Biology

Article Title: Mechanism of Action of a Distal NF-κB-Dependent Enhancer

doi: 10.1128/mcb.00271-06

Figure Lengend Snippet: FIG. 2. RelA and Sp1 are required for generation of the 3C product. (A) Murine cell lines deficient for RelA or Sp1 were compared to a wild-type MEF line for their ability to form the MCP-1 3C product. Cells were treated with TNF as indicated (, present; , absent), and formaldehyde was excluded in some experiments to control for nonspecific events. Only the wild-type (WT MEF) cells were able to generate a 3C assay product, suggesting that the 3C product is mediated by the factors bound to the DRR and PRR and that interactions between these regions occur in response to TNF. (B) Western blots for the presence of RelA, Sp1, or actin were conducted on the indicated cell lines. The lower migrating Sp1 band in the Sp1/ cells represents the N-terminal portion of Sp1 that is expressed in the knockout line. As described by Marin et al., this mutant is inactive in DNA binding and transactivation (38).

Article Snippet: The membrane was blocked with 10% nonfat dry milk and probed with either anti-CARM1 (Upstate Biotechnology, Inc.), anti-RelA/p65 (C-20, sc-372), anti-p300 (sc-585), anti-CBP (sc-369), antiSp1 (Sc-39), or anti-GAPDH antibodies (Santa Cruz Biotechnology, California) FIG. 1.

Techniques: Control, Western Blot, Knock-Out, Mutagenesis, Binding Assay

Journal: Molecular and Cellular Biology

Article Title: Mechanism of Action of a Distal NF-κB-Dependent Enhancer

doi: 10.1128/mcb.00271-06

Figure Lengend Snippet: FIG. 4. The Rel homology domain and serine 276 of RelA are required for expression and distal/proximal region interactions. A series of RelA mutants (A) were transfected into RelA-deficient cells and tested for their ability to drive expression of the endogenous gene (B) or an MCP-1 promoter CAT reporter gene (C) and for their ability to form the 3C product on the endogenous MCP-1 locus (D). TNF was not added to these assays, as overexpression of RelA is sufficient to bypass sequestration in the cytoplasm (48). All RelA expression constructions that contained a wild-type (WT) Rel homology domain were able to drive MCP-1 expression and generate a 3C product. (A) A schematic of the MCP-1 reporter gene construct (pJECAT2.6) and of the RelA p65 domain structure is shown. RHD, rel homology domain; TAD, transcriptional activation domain. Phosphorylatable serine residues shown to be important for RelA activity are indicated. (B) Real-time RT-PCR of MCP-1 mRNA levels from RelA-deficient cells transiently transfected as above with the indicated plasmids were normalized to the amount of GAPDH mRNA and plotted as the percentage of the wild-type RelA transfectant. The averages of results from three independent transfections are shown. (C) RelA-deficient cells transfected with the indicated RelA expression vector and the MCP-1 reporter. CAT values, derived from an enzyme-linked immunosorbent assay for CAT protein, normalized to the expression of an alkaline phosphatase expression vector were averaged from three experiments. (D) The 3C assay was carried out on the indicated plasmids transfected into the RelA-deficient cells as above. (E) Images from stained agarose gels representing three independent assays of the experiment shown in panel D were quantitated and plotted as described in Materials and Methods. (F) Western blots performed on cells transfected with the RelA mutant series show similar levels of expression between wild-type and mutant proteins. Different antibodies were used due to the loss of epitopes in some of the mutants (left blot, sc-372 [Santa Cruz, Inc.]; right blot, 12145-30 [Abcam, Inc.]).

Article Snippet: The membrane was blocked with 10% nonfat dry milk and probed with either anti-CARM1 (Upstate Biotechnology, Inc.), anti-RelA/p65 (C-20, sc-372), anti-p300 (sc-585), anti-CBP (sc-369), antiSp1 (Sc-39), or anti-GAPDH antibodies (Santa Cruz Biotechnology, California) FIG. 1.

Techniques: Expressing, Transfection, Over Expression, Construct, Activation Assay, Activity Assay, Quantitative RT-PCR, Plasmid Preparation, Derivative Assay, Enzyme-linked Immunosorbent Assay, Staining, Western Blot, Mutagenesis

Journal: Molecular and Cellular Biology

Article Title: Mechanism of Action of a Distal NF-κB-Dependent Enhancer

doi: 10.1128/mcb.00271-06

Figure Lengend Snippet: FIG. 5. CBP and p300 are required for MCP-1 expression and distal/proximal region interaction. siRNAs to CBP, p300, or both were transfected into NIH 3T3 cells. Cells were incubated for 48 h and then treated with TNF for 2 h. (A) Western blot analysis of the transfected cells using antibodies to CBP (-CBP), p300 (-p300), RelA (-p65), Sp1 (-Sp1), and GAPDH (-GAPDH), demonstrating specificity and efficacy of the siRNA. (B) In similar siRNA transfections, MCP-1 expression was measured by real-time RT-PCR. (C) The 3C assay was performed on the transfected cells treated with TNF as above. All siRNA transfections and assays were performed at least three times with identical results. , present; , absent.

Article Snippet: The membrane was blocked with 10% nonfat dry milk and probed with either anti-CARM1 (Upstate Biotechnology, Inc.), anti-RelA/p65 (C-20, sc-372), anti-p300 (sc-585), anti-CBP (sc-369), antiSp1 (Sc-39), or anti-GAPDH antibodies (Santa Cruz Biotechnology, California) FIG. 1.

Techniques: Expressing, Transfection, Incubation, Western Blot, Quantitative RT-PCR

Journal: Molecular and Cellular Biology

Article Title: Mechanism of Action of a Distal NF-κB-Dependent Enhancer

doi: 10.1128/mcb.00271-06

Figure Lengend Snippet: FIG. 6. CARM1 is required after the distal and proximal regions interact. CARM1-deficient MEFs were used to assay its role in MCP-1 activation. (A) Immunoblot analysis confirms that CARM1 protein is absent in the mutant cells and that CBP, p300, RelA (p65), Sp1, and GAPDH protein levels are unaffected. , anti. (B) CARM1-deficient cells fail to activate MCP-1 in response to TNF. Real-time RT-PCR was conducted on RNA samples that were isolated from wild-type (wt) and CARM1-deficient (m) MEFs following no TNF stimulation () or 2 h of TNF stimulation (). Real-time RT-PCR values were normal- ized to the amount of GAPDH mRNA in the samples and plotted with respect to the wild-type sample induced with TNF. The average of three independent experiments is shown. (C) An interaction forms between the proximal and distal regulatory regions regardless of whether CARM1 is present. The 3C assay as described above was performed on wild-type and CARM1-deficient (mut) MEFs. All sam- ples in this assay were derived from cells treated with TNF. All exper- iments were performed three times with identical results.

Article Snippet: The membrane was blocked with 10% nonfat dry milk and probed with either anti-CARM1 (Upstate Biotechnology, Inc.), anti-RelA/p65 (C-20, sc-372), anti-p300 (sc-585), anti-CBP (sc-369), antiSp1 (Sc-39), or anti-GAPDH antibodies (Santa Cruz Biotechnology, California) FIG. 1.

Techniques: Activation Assay, Western Blot, Mutagenesis, Quantitative RT-PCR, Isolation, Derivative Assay

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.

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: 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.

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: 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.

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

Journal: BMC Genomics

Article Title: RRAD, IL4I1, CDKN1A, and SERPINE1 genes are potentially co-regulated by NF-κB and p53 transcription factors in cells exposed to high doses of ionizing radiation

doi: 10.1186/s12864-018-5211-y

Figure Lengend Snippet: Activation of NF-κB and p53 pathways in U-2 OS cells exposed to a single high dose of ionizing radiation. a Kinetics of IR-induced nuclear translocation of RelA and p53 analyzed by Western blot in nuclear extracts (nuclear protein NPM3 was used as a loading reference). b Kinetics of RelA and p53 binding in the promoter of CXCL8 and CDKN1A gene, respectively, analyzed by ChIP-qPCR. c Kinetics of IR-induced activation of CXCL8 and CDKN1A gene expression analyzed by RT-qPCR. d Total levels of RelA and p53 proteins in cells transfected with specific siRNA, either untreated or 4 h. after 10 Gy irradiation, analyzed by Western blot in whole cell lysates

Article Snippet: Chromatin immunoprecipitation was carried out using the iDeal ChIP-seq Kit for Transcription Factors (Diagenode) with 3 μl/sample of anti-RelA(p65) polyclonal antibody (C15310256, Diagenode) or anti-p53 polyclonal antibody (C15410083, Diagenode), or without antibody (mock-IP), according to the manufacturer protocol.

Techniques: Activation Assay, Translocation Assay, Western Blot, Binding Assay, Expressing, Quantitative RT-PCR, Transfection, Irradiation

Journal: BMC Genomics

Article Title: RRAD, IL4I1, CDKN1A, and SERPINE1 genes are potentially co-regulated by NF-κB and p53 transcription factors in cells exposed to high doses of ionizing radiation

doi: 10.1186/s12864-018-5211-y

Figure Lengend Snippet: Detection of genes potentially regulated by radiation-activated p53 and NF-κB by integrative genomics. a Flowchart of the study. b Number of genes upregulated and downregulated by irradiation (at either 4 or 10 Gy dose) in wt cells, where IR induced or enhanced binding of either RelA or p53 in regulatory regions (listed are protein-coding genes with the binding of both transcription factors). c Number of genes with expression significantly affected by radiation (upregulated/downregulated, respectively; at either dose), whose expression was either inhibited or stimulated in cells with siRNA-silenced RELA or TP53 (listed are protein-coding genes whose IR-modulated expression was affected by silencing of both transcription factors). d Number of radiation-upregulated genes with IR-stimulated promoter binding of either RelA or p53 (in wt cells), which IR-modulated expression was affected in cells with siRNA-silenced RELA or TP53 (listed are “TF-dependent” genes, i.e., protein-coding genes with expression affected by silencing of transcription factor (TF) where radiation stimulated promoter binding of the corresponding TF)

Article Snippet: Chromatin immunoprecipitation was carried out using the iDeal ChIP-seq Kit for Transcription Factors (Diagenode) with 3 μl/sample of anti-RelA(p65) polyclonal antibody (C15310256, Diagenode) or anti-p53 polyclonal antibody (C15410083, Diagenode), or without antibody (mock-IP), according to the manufacturer protocol.

Techniques: Irradiation, Binding Assay, Expressing

Journal: BMC Genomics

Article Title: RRAD, IL4I1, CDKN1A, and SERPINE1 genes are potentially co-regulated by NF-κB and p53 transcription factors in cells exposed to high doses of ionizing radiation

doi: 10.1186/s12864-018-5211-y

Figure Lengend Snippet: Structure of κB and p53 binding motifs identified in analyzed genes. a The most probable common binding motifs revealed after analysis of ChIP-Seq data for all IR-induced peaks (±500 bp from the summits of p65 and p53 binding peaks). b Sequence and localization of the most probable motifs in the nearest vicinity of selected genes. Presented are binding regions identified by ChiP-Seq (upper inserts) and localization of analyzed PCR products (red and green bars) together with corresponding gene structures (exons and UTRs are marked with wide and narrow blue boxes, respectively); shown are distances from the Transcription Start Sites of the major transcript variants; red and green marks were used for RelA (p65) and p53, respectively

Article Snippet: Chromatin immunoprecipitation was carried out using the iDeal ChIP-seq Kit for Transcription Factors (Diagenode) with 3 μl/sample of anti-RelA(p65) polyclonal antibody (C15310256, Diagenode) or anti-p53 polyclonal antibody (C15410083, Diagenode), or without antibody (mock-IP), according to the manufacturer protocol.

Techniques: Binding Assay, ChIP-sequencing, Sequencing

Journal: BMC Genomics

Article Title: RRAD, IL4I1, CDKN1A, and SERPINE1 genes are potentially co-regulated by NF-κB and p53 transcription factors in cells exposed to high doses of ionizing radiation

doi: 10.1186/s12864-018-5211-y

Figure Lengend Snippet: Genes putatively co-regulated by NF-κB and p53; validation of genomics profiling. a The influence of silencing of RELA and TP53 on gene expression analyzed by RT-qPCR in the same set of genes 4 h after irradiation with 10 Gy. Expression levels are presented as a fold-change vs. untreated wild-type control cells (dots represent mean values); asterisks indicate statistical significance of differences between cells treated with control siRNA and cells treated with siRNA specific for RELA or TP53 : * p < 0.05, ** p < 0.001. b Radiation-stimulated binding of RelA (p65) and p53 in putative regulatory regions of selected genes analyzed by ChIP-qPCR; p53 binding was additionally illustrated by gel electrophoresis of PCR products (C – control, 2 and 4–2 and 4 h after irradiation with 10 Gy). Marked are median, maximum and minimum values of a fold-change vs. untreated control (asterisks indicate statistical significance of differences: * p < 0.05, ** p < 0.001)

Article Snippet: Chromatin immunoprecipitation was carried out using the iDeal ChIP-seq Kit for Transcription Factors (Diagenode) with 3 μl/sample of anti-RelA(p65) polyclonal antibody (C15310256, Diagenode) or anti-p53 polyclonal antibody (C15410083, Diagenode), or without antibody (mock-IP), according to the manufacturer protocol.

Techniques: Expressing, Quantitative RT-PCR, Irradiation, Binding Assay, Nucleic Acid Electrophoresis

Journal: Nucleic Acids Research

Article Title: NF-κB factors cooperate with Su(Hw)/E4F1 to balance Drosophila /human immune responses via modulating dynamic expression of miR-210

doi: 10.1093/nar/gkae394

Figure Lengend Snippet: hsa-miR-210 directly targets TLR6 and RelA-mediated transcriptional repression of hsa-miR-210 depends on E4F1. ( A ) Potential binding sites of hsa-miR-210 in 3'UTR region of TLR6 were predicted using the miRWalk website. Point mutations were introduced at the target site in the 3'UTR, which resulted in base-pairing with the seed sequence of hsa-miR-210 . ( B ) Dual luciferase assay was performed in 293T cells to determine the luciferase activities of mimics- hsa-miR-210 and a reporter plasmid with or without the mutation site of target TLR6 . ( C ) RIP-qPCR experiment was performed in THP1-induced macrophages transfected with mimics-NC or mimics- hsa-miR-210 , using anti-Ago2 antibody. ( D ) Schematic diagram illustrates the promoter region of hsa-miR-210 . The predicted TSS, position of the designed primer (purple line), RelA motifs (blue line) and E4F1 motifs (orange line) were marked. ( E ) Luciferase assay was performed in 293T cells to determine the activity of pGL3-hsa-miR-210-promoter and the pGL3-hsa-miR-210-promoter-AS reporters. ( F ) Luciferase assay was performed in 293T cells to determine the activity of the pGL3- hsa-miR-210 -promoter reporters. Exogenous RelA and E4F1 were separately or jointly included in the assay. “+” indicated transfection with 500 ng of this plasmid, “–” indicated no transfection of the plasmid in 293T cells. ( G ) Luciferase assay was performed in 293T cells to determine the activity of the pGL3- hsa-miR-210 -promoter-RelA-motif-mut reporters. Exogenous RelA and E4F1 were separately or jointly included in the assay. “+” indicated transfection with 500 ng of this plasmid, “–” indicated no transfection of the plasmid in 293T cells. ( H ) Luciferase assay was performed in 293T cells to determine the activity of the pGL3- hsa-miR-210 -promoter-E4F1-motif-mut reporters. Exogenous RelA and E4F1 were separately or jointly included in the assay. “+” indicated transfection with 500 ng of this plasmid, “–” indicated no transfection of the plasmid in 293T cells.

Article Snippet: In the Co-IP procedure, 2 × 10 7 cells or five flies were collected and suspended in RIPA buffer supplemented with protease inhibitor cocktail (Roche, USA) and PMSF on ice for 30 min. After centrifuging at 13 000 rpm for 10 min at 4°C, the lysates (equal allocation for each IP and Input experiment) were incubated with anti-V5 tag antibody (ABclonal, #AE017, China), anti-Flag tag antibody (ABclonal, #AE005, China), anti-HA tag antibody (ABclonal, #AE008), anti-RelA Rabbit mAb (ABclonal #A19653, China), anti-E4F1 (D-12) monoclonal antibody (SANTA # sc-514718, USA), anti-GFP antibody (abcam, #ab290, USA) and anti-Dorsal 7A4 (DSHB # AB_528204, USA) at 4°C overnight, followed by further incubation with Protein A/G PLUS-Agarose (SANTA,# sc-2003) for 2 h. The beads were washed five times with PBST buffer.

Techniques: Binding Assay, Sequencing, Luciferase, Plasmid Preparation, Mutagenesis, Transfection, Activity Assay

Journal: Nucleic Acids Research

Article Title: NF-κB factors cooperate with Su(Hw)/E4F1 to balance Drosophila /human immune responses via modulating dynamic expression of miR-210

doi: 10.1093/nar/gkae394

Figure Lengend Snippet: Interaction between RelA and E4F1 suppresses hsa-miR-210 transcription in the early stage of G+ bacterial stimulation. ( A ) In vitro Co-IP experiments were performed in 293T cells co-transfected with Flag-RelA and HA-E4F1 using anti-Flag and anti-HA antibodies for immunoprecipitation, followed by western blot analysis using corresponding antibodies. ( B ) In vivo Co-IP experiments were performed in THP1-induced macrophages, both uninfected and at 0.5 h post-infection, using anti-RelA, anti-E4F1 antibodies for immunoprecipitation, followed by western blot analysis using corresponding antibodies. ( C ) ChIP-qPCR was performed in THP1-induced macrophages, with and without overexpression of RelA at 0.5 h post-infection, to detect fold change on the promoters of hsa-miR-210 using anti-RelA antibodies for the immunoprecipitation in vivo . ( D ) ChIP-qPCR was performed in THP1-induced macrophages, with and without overexpression of RelA at 0.5 h post-infection, to detect fold change on the promoters of hsa-miR-210 using anti-E4F1 antibodies for the immunoprecipitation in vivo . ( E ) ChIP-qPCR was performed in THP1-induced macrophages, with and without overexpression of E4F1 at 0.5 h post-infection, to detect fold change on the promoters of hsa-miR-210 using anti-E4F1 antibodies for the immunoprecipitation in vivo . ( F ) ChIP-qPCR was performed in THP1-induced macrophages, with and without overexpression of E4F1 at 0.5 h post-infection, to detect fold change on the promoters of hsa-miR-210 using anti-RelA antibodies for the immunoprecipitation in vivo .

Article Snippet: In the Co-IP procedure, 2 × 10 7 cells or five flies were collected and suspended in RIPA buffer supplemented with protease inhibitor cocktail (Roche, USA) and PMSF on ice for 30 min. After centrifuging at 13 000 rpm for 10 min at 4°C, the lysates (equal allocation for each IP and Input experiment) were incubated with anti-V5 tag antibody (ABclonal, #AE017, China), anti-Flag tag antibody (ABclonal, #AE005, China), anti-HA tag antibody (ABclonal, #AE008), anti-RelA Rabbit mAb (ABclonal #A19653, China), anti-E4F1 (D-12) monoclonal antibody (SANTA # sc-514718, USA), anti-GFP antibody (abcam, #ab290, USA) and anti-Dorsal 7A4 (DSHB # AB_528204, USA) at 4°C overnight, followed by further incubation with Protein A/G PLUS-Agarose (SANTA,# sc-2003) for 2 h. The beads were washed five times with PBST buffer.

Techniques: In Vitro, Co-Immunoprecipitation Assay, Transfection, Immunoprecipitation, Western Blot, In Vivo, Infection, ChIP-qPCR, Over Expression

Journal: Nucleic Acids Research

Article Title: NF-κB factors cooperate with Su(Hw)/E4F1 to balance Drosophila /human immune responses via modulating dynamic expression of miR-210

doi: 10.1093/nar/gkae394

Figure Lengend Snippet: The RelA/E4F1/ hsa-miR-210 / TLR6 regulation loop contributes to innate immune responses in THP1-induced macrophages. ( A ) RT-qPCR analysis of IFNB , IL6 and TNFA expression was measured in THP1-induced macrophages transfected with mimics-NC or mimics- hsa-miR-210 and stimulated with heat-killed M. luteus [multiplicity of infection (MOI) = 50]. ( B ) RT-qPCR analysis of TLR6 expression was measured in THP1-induced macrophages transfected with mimics-NC or mimics- hsa-miR-210 and stimulated with heat-killed M. luteus [multiplicity of infection (MOI) = 50]. ( C ) Changes in the protein levels of TLR6 were assessed in THP1-induced macrophages with mimics-NC or mimics- hsa-miR-210 transfection and heat-killed M. luteus [multiplicity of infection (MOI) = 50] stimulated. ( D ) Changes in the protein levels of E4F1, Phospho-RelA, RelA and GAPDH were analyzed in THP1-induced macrophages using western blot at different time points post-infection. ( E ) Western blot grayscale analysis of E4F1 in THP1-induced macrophages at different time points post-infection. ( F ) Western blot grayscale analysis of Phospho-RelA in THP1-induced macrophages at different time points post-infection. ( G ) The dynamic expressions of hsa-miR-210 in THP1-induced macrophages transfected with inhibitor-NC and inhibitor- hsa-miR-210 were detected by RT-qPCR at 0, 0.25, 0.5, 1, 2, 3 h post-infection. ( H ) The dynamic expressions of TLR6 in THP1-induced macrophages transfected with inhibitor-NC and inhibitor- hsa-miR-210 were detected by RT-qPCR at 0, 0.25, 0.5, 1, 2, 3 h post-infection.

Article Snippet: In the Co-IP procedure, 2 × 10 7 cells or five flies were collected and suspended in RIPA buffer supplemented with protease inhibitor cocktail (Roche, USA) and PMSF on ice for 30 min. After centrifuging at 13 000 rpm for 10 min at 4°C, the lysates (equal allocation for each IP and Input experiment) were incubated with anti-V5 tag antibody (ABclonal, #AE017, China), anti-Flag tag antibody (ABclonal, #AE005, China), anti-HA tag antibody (ABclonal, #AE008), anti-RelA Rabbit mAb (ABclonal #A19653, China), anti-E4F1 (D-12) monoclonal antibody (SANTA # sc-514718, USA), anti-GFP antibody (abcam, #ab290, USA) and anti-Dorsal 7A4 (DSHB # AB_528204, USA) at 4°C overnight, followed by further incubation with Protein A/G PLUS-Agarose (SANTA,# sc-2003) for 2 h. The beads were washed five times with PBST buffer.

Techniques: Quantitative RT-PCR, Expressing, Transfection, Infection, Western Blot