Journal: International Journal of Molecular Sciences

Article Title: Resveratrol Modulates Chemosensitisation to 5-FU via β1-Integrin/HIF-1α Axis in CRC Tumor Microenvironment

doi: 10.3390/ijms24054988

Figure Lengend Snippet: Resveratrol’s reduction of inflammation, vascularisation as well as cancer stemness and elevation of apoptosis via β1-integrin receptors in HCT-116/HCT-116R cells shown by Western blot analysis. X -axis: HCT-116 ( A ) and HCT-116R ( B ) cells in alginate drops were left untreated alone (Co.) or in TME, where they were left untreated or were treated with 2 nM 5-FU, 5 µM resveratrol, 0.5 µM β1-SO, 0.5 µM β1-ASO or combinations thereof. Samples were immunoblotted with antibodies against NF-kB (unphosphorylated NF-kB), p-NF-kB (phosphorylated NF-kB), cleaved-caspase-3, HIF-1α, VEGF, CD44, CD133, ALDH1 and β-actin (loading control). Y -axis shows densitometric units. Relative to TME control, values were p < 0.05 (⋆) and p < 0.01 (⋆⋆).

Article Snippet: The monoclonal antibodies against phospho-p65-NF-kB (#MAB7226), p65-NF-kB (#MAB5078) as well as polyclonal anti-cleaved-caspase-3 (#AF835) were acquired from R&D Systems (Heidelberg, Germany), while monoclonal antibody against β1-integrin (#14-0299-82) was from Thermo Fisher Scientific (Langenselbold, Germany).

Techniques: Western Blot, Control

Journal: Molecular Medicine Reports

Article Title: Buyang Huanwu Tang improves denervation-dependent muscle atrophy by increasing ANGPTL4, and increases NF-κB and MURF1 levels

doi: 10.3892/mmr.2017.8306

Figure Lengend Snippet: Examination of NF-κB p65 and MURF1 protein expression in Model and BYHWT treatment group. (A) Western blot assay for NF-κB p65 and MURF1 expression. (B) Semi-quantification of protein expression. *P<0.05 vs. normal control group. # P<0.05 vs. model group. BYHWT, Buyang Huanwu Tang; NF-κB, nuclear factor-κB; MURF1, muscle RING-finger protein-1.

Article Snippet: The membrane was blocked by using 5% non-fat milk at room temperature for 2 h, followed by incubation with the rabbit anti-rat ANGPTL4 polyclonal antibody (1:2,000; cat. no. AF3485; R&D Systems, Inc., Minneapolis, MN, USA), rabbit anti-rat NF-κB p65 polyclonal antibody (1:1,000; cat. no. MAB50781; R&D Systems, Inc.), mouse anti-rat MURF1 monoclonal antibody (1:3,000; cat. no. AF5366; R&D Systems, Inc.) and the rabbit anti-rat actin (internal control, cat. no. MAB1420; R&D Systems, Inc.) polyclonal antibody at 4°C overnight.

Techniques: Expressing, Western Blot, Control

Journal: Frontiers in Aging Neuroscience

Article Title: Adenosine A 2A Receptor in Bone Marrow-Derived Cells Mediated Macrophages M2 Polarization via PPARγ-P65 Pathway in Chronic Hypoperfusion Situation

doi: 10.3389/fnagi.2021.792733

Figure Lengend Snippet: The expression of PPARγ, P65, and p-P65 was altered in macrophages cultured in low glucose and hypoxia. (A) Representative electrophoretic bands showing the expression of PPARγ, P65 and p-P65 in control, CGS21680 and SCH58261 treated macrophages at post-culture. β-actin was used as internal control. (B) Statistical analysis showing the expression of PPARγ in macrophages was increased gradually in low glucose and hypoxic conditions, and was increased further by CGS21680 at post-culture 12 h, but SCH58261 reduced the expression of PPARγ at post-culture (ns: P > 0.05, *** P < 0.001. Two-way ANOVA test followed by Turkey's multiple comparisons test. n = 3 independent experiments for each group). (C) Statistical analysis showing the expression of P65 was increased in macrophages at post-culture 12 and 24 h, and the expression was further increased by CGS21680 at 12 h and SCH58261 at 6 and 12 h (ns: P > 0.05, *** P < 0.001. Two-way ANOVA test followed by Turkey's multiple comparisons test. n = 3 independent experiments for each group). (D) Statistical analysis showing the expression of p-P65 was increased by CGS21680 at post-culture 6 and 24 h, and was increased in SCH58261 treated macrophages at post-culture 6, 12, and 24 h (* P < 0.05, *** P < 0.001. Two-way ANOVA test followed by Turkey's multiple comparisons test. n = 3 independent experiments for each group). (E,F) Statistical analysis showing the mRNA of PPAR γ was increased in CGS21680 treated macrophages at 2 and 12 h, but not affected by SCH58261. The expression of P65 mRNA was increased in SCH58261 treated macrophages at post-culture 12 h (ns: P > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001. Two-way ANOVA test followed by Turkey's multiple comparisons test. n = 3 independent experiments for each group). ns, non significant; p >0.05.

Article Snippet: The membranes were blocked by 5% skimmed milk and incubated overnight at 4°C with the following primary antibodies: a rabbit anti-PPARγ antibody (1:1000, AF6284, Affinity Bioscience, Beijing, China), rabbit anti-P65 antibody (1:1000, NB100-2176, NOVUS Biologicals, Building IV Centennial, CO 80112, USA), or rabbit anti-p-P65 antibody (1:1000, NB100-82088, NOVUS Biologicals, Building IV Centennial, CO 80112, USA).

Techniques: Expressing, Cell Culture, Control

Journal: Frontiers in Aging Neuroscience

Article Title: Adenosine A 2A Receptor in Bone Marrow-Derived Cells Mediated Macrophages M2 Polarization via PPARγ-P65 Pathway in Chronic Hypoperfusion Situation

doi: 10.3389/fnagi.2021.792733

Figure Lengend Snippet: PPARγ regulated the expression of P65 and p-P65 in CGS21680 treated macrophages. (A) Representative electrophoretic bands showing the expression of P65 and p-P65 in PPARγ shRNA pre-transfected macrophages with or without CGS21680 treatment after cultured in low glucose and hypoxic conditions. (B,C) Densitometric analysis results showing the expression of P65 were reduced in shRNA targeted macrophages and further reduced by CGS21680 at post-culture 12 h (* P < 0.05, *** P < 0.001. Two-way ANOVA test followed by Turkey's multiple comparisons test. n = 3 independent experiments for each group). (C) The ratio of p-P65/P65 was increased in macrophages upon PPARγ knockdown at post-culture 12 and 24 h, but was reduced in by additional CGS21680 treatment at post-culture 2 and 12 h (*** P < 0.001. Two-way ANOVA test followed by Turkey's multiple comparisons test. n = 3 independent experiments for each group). (D) RT-PCR results showing the mRNA expression of P65 was reduced in macrophages upon PPARγ knockdown at post-culture 12 h and was potentiated by additional CGS21680 treatment at post-culture 6 and 12 h (* P < 0.05, *** P < 0.001. Two-way ANOVA test followed by Turkey's multiple comparisons test. n = 3 independent experiments for each group). ** P < 0.01.

Article Snippet: The membranes were blocked by 5% skimmed milk and incubated overnight at 4°C with the following primary antibodies: a rabbit anti-PPARγ antibody (1:1000, AF6284, Affinity Bioscience, Beijing, China), rabbit anti-P65 antibody (1:1000, NB100-2176, NOVUS Biologicals, Building IV Centennial, CO 80112, USA), or rabbit anti-p-P65 antibody (1:1000, NB100-82088, NOVUS Biologicals, Building IV Centennial, CO 80112, USA).

Techniques: Expressing, shRNA, Transfection, Cell Culture, Knockdown, Reverse Transcription Polymerase Chain Reaction

Journal: Molecular and Cellular Biology

Article Title: Functional Interplay between Acetylation and Methylation of the RelA Subunit of NF-κB

doi: 10.1128/mcb.01343-09

Figure Lengend Snippet: FIG. 1. Set9 methylates lysines 314 and 315 on promoters of NF-B target genes. (A) Recombinant proteins of WT RelA or RelA-K314/ 315R were incubated with recombinant WT Set9 or Set9-H297A in an in vitro methylation assay. Reaction products were separated by SDS- polyacrylamide gel electrophoresis, and methylation was detected by immunoblotting (IB) with anti-methylated lysine 314/315 antibodies (top). Levels of RelA, RelA-K314/315R, Set9, and Set9-H297A are shown at the bottom. (B) HEK293T cells were cotransfected with expression vectors encoding WT RelA or RelA-K314/315R and Set9 or Set9-H297A, as indicated. Methylation was detected by immuno- blotting of anti-T7 immunoprecipitates (IP) with anti-methylated ly- sine 314/315 antibodies. Levels of T7-RelA, RelA-K314/315R, Set9, and Set9-H297A are shown at the bottom. (C) U2OS cells were trans- fected with control (Ctr) siRNA or Set9 siRNA and stimulated with TNF- as indicated. Methylation of RelA was assessed by immuno- blotting the anti-Me-K314/315 RelA immunoprecipitates with RelA antibody. (D) RelA-deficient MEFs reconstituted with empty vector, WT RelA, or RelA-K314/315R were untreated or stimulated with TNF- (20 ng/ml) for 30 min, and RelA methylation was assessed as described above (C). (E) U2OS cells were stimulated with TNF- as

Article Snippet: Polyclonal antibodies against monomethylated lysine 314/315 RelA (anti-MeK314/315) were generated by New England Peptide with a synthesized peptide corresponding to amino acids (aa) 308 to 320 of RelA (NH2-TFKSIMK[Me]K [Me]SPFSGC-COOH) as the antigen.

Techniques: Recombinant, Incubation, In Vitro, Methylation, Polyacrylamide Gel Electrophoresis, Western Blot, Expressing, Control, Plasmid Preparation

Journal: Molecular and Cellular Biology

Article Title: Functional Interplay between Acetylation and Methylation of the RelA Subunit of NF-κB

doi: 10.1128/mcb.01343-09

Figure Lengend Snippet: FIG. 2. Acetylation of RelA at lysine 310 inhibits methylation of RelA at lysines 314 and 315 in vitro. (A) Schematic domain structure of RelA and the sequences surrounding the acetylation site (lysine 310) and methylation sites (lysines 314 and 315). NLS, nuclear localization signal. RHD, Rel homology domain; TA, transactivation domain. (B) RelA peptides (aa 308 to 320) unmodified (UM) or methylated (Me) at lysines 314 and 315 were in vitro acetylated by p300 in the presence of [14C]acetyl coenzyme A (CoA). Samples were separated in a Tris-Tricine gel, and acetylation levels and peptide loading amounts were assessed by autoradiography and silver staining, respectively. (C) RelA peptides (aa 305 to 324) unmodified (UA) or acetylated (Ac) at lysine 310 were in vitro methylated by Set9 in the presence of [3H]S-adenosine-methionine (SAM). Samples were separated in a Tris-Tricine gel, and methylation levels and peptide loading amounts were assessed as described above (B). (D) Full-length RelA was in vitro acetylated by p300 in the absence or presence of acetyl-CoA and then subjected to a Set9-mediated in vitro methylation assay in the presence of [3H]SAM. Samples were separated by SDS-PAGE, and levels of methylated RelA were assessed by either autoradiography or immunoblotting with anti-methylated lysine 314/315 antibodies. Levels of acetylated RelA and total RelA are shown at the bottom. (E) Unmodified or in vitro-methylated recombinant full-length RelA was subjected to a p300-mediated in vitro acetylation assay. Samples were separated by SDS-PAGE; levels of acetylated RelA, methylated RelA, or total RelA were assessed by immunoblotting as indicated. (F) A series of dilutions of RelA peptides (aa 308 to 320), including unmodified, monomethylated at lysines 314 and 315 (Me-K314/315), or acetylated at lysine 310 and monomethylated at lysines 314 and 315 (Ac-K310/Me-K314/315), were spotted onto a nitrocellulose membrane. Dot blot analysis was performed with antibodies as indicated. The Ponceau S staining of the same membrane served as the peptide loading control.

Article Snippet: Polyclonal antibodies against monomethylated lysine 314/315 RelA (anti-MeK314/315) were generated by New England Peptide with a synthesized peptide corresponding to amino acids (aa) 308 to 320 of RelA (NH2-TFKSIMK[Me]K [Me]SPFSGC-COOH) as the antigen.

Techniques: Methylation, In Vitro, Autoradiography, Silver Staining, SDS Page, Western Blot, Recombinant, Acetylation Assay, Membrane, Dot Blot, Staining, Control

Journal: Molecular and Cellular Biology

Article Title: Functional Interplay between Acetylation and Methylation of the RelA Subunit of NF-κB

doi: 10.1128/mcb.01343-09

Figure Lengend Snippet: FIG. 3. Acetylation at lysine 310 inhibits methylation of RelA in vivo. (A) HEK293T cells were transfected with empty vector or ex- pression vectors for p300 or the p300-HAT mutant. At 30 h post- transfection, cells were treated with TNF- for the indicated time periods. Levels of methylated or acetylated RelA were assessed by immunoblotting anti-methylated lysine 314/315 or anti-acetylated ly- sine 310 immunoprecipitates, respectively, with RelA antibodies. The quantitation of methylated or acetylated RelA (fold increase or de- crease relative to levels of RelA shown at the bottom) is indicated below each sample. (B) HEK293T cells were transfected with an empty vector or expression vectors for Flag-SIRT1 or the Flag-SIRT1 (H363Y) mutant. At 30 h posttransfection, cells were treated with TNF- for the indicated time periods. Levels of methylated or acety- lated RelA were assessed as described above (A). (C) HEK293T cells were transfected with an empty vector or expression vectors for SIRT1 shRNA. TNF--induced RelA methylation at K314/315 and acetyla- tion at K310 were assessed as described above (A). Levels of RelA and

Article Snippet: Polyclonal antibodies against monomethylated lysine 314/315 RelA (anti-MeK314/315) were generated by New England Peptide with a synthesized peptide corresponding to amino acids (aa) 308 to 320 of RelA (NH2-TFKSIMK[Me]K [Me]SPFSGC-COOH) as the antigen.

Techniques: Methylation, In Vivo, Transfection, Plasmid Preparation, Mutagenesis, Western Blot, Quantitation Assay, Expressing, shRNA

Journal: Molecular and Cellular Biology

Article Title: Functional Interplay between Acetylation and Methylation of the RelA Subunit of NF-κB

doi: 10.1128/mcb.01343-09

Figure Lengend Snippet: FIG. 4. Mutation of lysine 310 regulates methylation of RelA in vivo. (A) HEK293T cells were transfected with expression vectors for T7-tagged WT RelA, RelA-K310Q, or RelA-K310R together with green fluorescent protein (GFP)-Set9. The methylation of RelA was assessed as described in the legend of Fig. 1B. RelA-associated Set9 was detected by immunoblotting of the RelA immunoprecipitates with anti-GFP antibodies. Levels of RelA and Set9 are shown in the bottom two panels. The quantitation of methylated RelA (fold increase or decrease relative to levels of RelA shown at the bottom) is indicated below each sample. (B) RelA-deficient MEFs reconstituted with WT RelA, RelA-K310Q, or RelA-K310R were stimulated with TNF- as indicated. Levels of methylated RelA were determined as described in the legend of Fig. 1C. The quantitation of methylated RelA (fold increase or decrease relative to levels of RelA shown at the bottom) is indicated below each sample. (C) RelA-deficient MEFs reconstituted with WT RelA, RelA-K310Q, or RelA-K310R were transfected with 5 B and Renilla luciferase reporter plasmids and stimulated with TNF- 24 h after transfection. Luciferase activity was measured 5 h after stimulation. Results represent the averages of data from three independent experiments SD. *, P 0.05.

Article Snippet: Polyclonal antibodies against monomethylated lysine 314/315 RelA (anti-MeK314/315) were generated by New England Peptide with a synthesized peptide corresponding to amino acids (aa) 308 to 320 of RelA (NH2-TFKSIMK[Me]K [Me]SPFSGC-COOH) as the antigen.

Techniques: Mutagenesis, Methylation, In Vivo, Transfection, Expressing, Western Blot, Quantitation Assay, Luciferase, Activity Assay

Journal: Molecular and Cellular Biology

Article Title: Functional Interplay between Acetylation and Methylation of the RelA Subunit of NF-κB

doi: 10.1128/mcb.01343-09

Figure Lengend Snippet: FIG. 5. Acetylation regulates the ubiquitination and stability of RelA. (A) WT or SIRT1-deficient MEFs were pulse stimulated with TNF- for 15 min, followed by treatment with the proteasome inhibitor MG132 for 4 h. Levels of ubiquitinated RelA were assessed by immunoblotting the RelA immunoprecipitates prepared from chromatin-associated proteins with antiubiquitin (Ubn) antibodies. Levels of RelA, SIRT1, and tubulin are shown at the bottom. The quantitation of ubiquitinated RelA (fold increase or decrease relative to levels of RelA shown at the bottom) is indicated below each sample. (B) TNF--induced ubiquitination of RelA in RelA-deficient MEFs reconstituted with WT RelA, RelA-K310Q, or RelA-K310R was measured and quantitated as described above (A). Levels of RelA from different cells are shown at the bottom. (C) WT or SIRT1-deficient MEFs were pulse stimulated with TNF- for 15 min, followed by a chase in the presence of 10 g/ml cycloheximide (CHX). Cells were harvested at the indicated time points, and chromatin-associated proteins were extracted and immunoblotted for RelA. A typical result is shown at the top. The quantification of the results from three independent experiments are shown as means SD at the bottom. (D) Stability of RelA from RelA-deficient MEFs reconstituted with WT RelA, RelA-K310Q, or RelA-K310R was assessed as described above (C).

Article Snippet: Polyclonal antibodies against monomethylated lysine 314/315 RelA (anti-MeK314/315) were generated by New England Peptide with a synthesized peptide corresponding to amino acids (aa) 308 to 320 of RelA (NH2-TFKSIMK[Me]K [Me]SPFSGC-COOH) as the antigen.

Techniques: Ubiquitin Proteomics, Western Blot, Quantitation Assay

Journal: Molecular and Cellular Biology

Article Title: Functional Interplay between Acetylation and Methylation of the RelA Subunit of NF-κB

doi: 10.1128/mcb.01343-09

Figure Lengend Snippet: FIG. 6. Acetylation of lysine 310 of RelA inhibits its binding to Set9. (A) RelA peptides (aa 308 to 324) that were unmodified (unacetylated [UA])or acetylated at lysine 310 were incubated with recombinant His-tagged Set9 for 30 min. Set9 was recovered with Ni-NTA agarose beads and assessed by immunoblotting. Set9-associated peptides were separated in a Tris-Tricine gel, followed by silver staining. (B) WT RelA-, RelA- K310Q-, or RelA-K310R-reconstituted MEFs were left untreated or stimulated with TNF- for 30 min. Set9 immunoprecipitates prepared from whole-cell lysates were immunoblotted for RelA. Levels of RelA and Set9 are shown in the bottom two panels. (C) Structural model of the complex of the RelA peptide and Set9 constructed using complexes of Set9 with p53 and Set9 with ER. (Left) The enzyme is shown in a surface representation with the residues in red (acidic), blue (basic), green (polar, noncharged), and white (nonpolar). The backbone of the peptide (common for RelA and “the other peptide”) and the side chains of key residues in the model are shown explicitly and labeled. The methylated lysines (lysines 314 and 315 in RelA and lysine 302 in ER) are shown in a stick representation and are colored based on atom types (carbon in cyan and nitrogen in blue). The model shows how Lys310 in RelA can occupy the same position as Arg300 in ER (shown using a transparent stick representation), which is clamped by several negatively charged residues (red patches) from the Set9 enzyme. (Top right) Slightly rotated view of the two peptides (at left) with the enzyme removed in order to clearly demonstrate the occupancy of the same location by the positively charged moieties of ER/Arg300 and RelA/Lys310. (Bottom right) Sequence alignment of the p53, ER, and RelA peptides, with the lysine pair in green and the basic residues that interact with the negative clamp of the enzyme in magenta. (D) Sequence alignment of peptides from six Set9 substrates, with the methylation sites in green and the basic residues that might interact with the negative clamp of Set9 in red. The methylation site in each substrate is assigned a position of 0.

Article Snippet: Polyclonal antibodies against monomethylated lysine 314/315 RelA (anti-MeK314/315) were generated by New England Peptide with a synthesized peptide corresponding to amino acids (aa) 308 to 320 of RelA (NH2-TFKSIMK[Me]K [Me]SPFSGC-COOH) as the antigen.

Techniques: Binding Assay, Incubation, Recombinant, Western Blot, Silver Staining, Construct, Labeling, Methylation, Sequencing

Journal: Molecular and Cellular Biology

Article Title: Functional Interplay between Acetylation and Methylation of the RelA Subunit of NF-κB

doi: 10.1128/mcb.01343-09

Figure Lengend Snippet: FIG. 7. Schematic model for the inhibition of RelA methylation by acetylation. The p300-mediated acetylation of lysine 310, which neu- tralizes the positive charge of this residue, inhibits the binding of Set9 to RelA, thereby impairing the Set9-mediated methylation of lysines 314 and 315. The deacetylation of RelA by SIRT1 restores the positive charge of lysine 310, which facilitates the recruitment of Set9 to RelA via a salt bridge interaction between the basic side chain of lysine 310 and the negatively charged exosite of Set9 and consequently leads to the methylation of lysines 314 and 315.

Article Snippet: Polyclonal antibodies against monomethylated lysine 314/315 RelA (anti-MeK314/315) were generated by New England Peptide with a synthesized peptide corresponding to amino acids (aa) 308 to 320 of RelA (NH2-TFKSIMK[Me]K [Me]SPFSGC-COOH) as the antigen.

Techniques: Inhibition, Methylation, Residue, Binding Assay

Journal: Cancers

Article Title: Transfection with GLS2 Glutaminase (GAB) Sensitizes Human Glioblastoma Cell Lines to Oxidative Stress by a Common Mechanism Involving Suppression of the PI3K/AKT Pathway

doi: 10.3390/cancers11010115

Figure Lengend Snippet: Transfection with GAB increases the sensitivity of GBM cell lines T98G, U87MG, and LN229 to H 2 O 2 -induced oxidative stress. Upon H 2 O 2 treatment, the GAB-transfected cells of all three cell lines present decreased levels of phosphorylated PI3K and PDK1 compared to the pcDNA-transfected counterparts. Hypothetically, changes in the activity of the PI3K signaling cascade may ensue modulation of RTKs by GAB, but experimental evidence to support this notion is absent. GAB-transfected T98G and U87MG cells display decreased AKT phosphorylation on both Thr308 and Ser473, reduced NF-κB phosphorylation, and increased activity of caspase 3 and 7, suggesting that caspase dependent apoptosis contributes to their death. By contrast, in the LN229 cell line, the lack of changes in the phosphorylation level of NF-κB and in caspase 3 and 7 activities indicates that the mechanism underlying GAB-mediated death is other than caspase dependent apoptosis. Note that in contrast to GAB-transfected T98G and U87MG cells, GAB-transfected LN229 cells present increased AKT phosphorylation on Ser473: The implications (if any) of this difference for the nature of cell death are not known.

Article Snippet: The following antibodies were used: p-AKT (Thr308) (#4056, Cell Signaling, Danvers, MA, USA), p-AKT (Ser473) (#4060, Cell Signaling), AKT (#4691, Cell Signaling), p-PDK1 (Ser241) (#ab109460, Abcam), PDK1 (#17086-1-AP, ProteinTech, Chicago, IL, USA), p-PI3K (Tyr199) (#4228, Cell signaling), PI3K (#11889, Cell Signaling), p-NF-κB (Ser536) (#MAB72261, Novus Biologicals, Littleton, CO, USA), and NF-κB (#4764, Cell Signaling) according to the manufacturer’s instruction.

Techniques: Transfection, Activity Assay, Phospho-proteomics

Journal: Journal of Clinical Investigation

Article Title: TET repression and increased DNMT activity synergistically induce aberrant DNA methylation

doi: 10.1172/jci124070

Figure Lengend Snippet: Figure 3. Increased RELA binding levels at pro- moter regions of TET-targeting miRNAs. (A) Acti- vation of NF-κB signaling pathway in NUGC-3 cells by TNF-α. A downstream target gene of NF-κB signaling pathway, IL6, was upregulated by TNF-α treatment. Data represent mean ± SE. (B) Heat- map of RELA binding levels in NUGC-3 cells treated with TNF-α. RELA binding levels at genomic regions around TSSs of 44,112 transcripts were aligned according to the binding level after TNF-α treatment. Clear increase by the treatment was observed. Each row shows ± 2.5 kb centered on TSS. (C) Enriched motifs in RELA peaks detected in NUGC-3 cells treated with TNF-α. NF-κB binding motifs were most significantly enriched in NUGC-3 cells treated with TNF-α, showing successful detection of RELA binding sites. (D–F) RELA bind- ing status around the putative promoter regions of TET-targeting miRNAs. RELA binding levels at putative promoter regions of MIR26B (CTDSP1) (D) and MIR20A (MIR17HG) (E) were robustly increased by TNF-α treatment. RELA binding levels at these host genes were comparable to that at the IL6 promoter (F). Black boxes indicate genomic regions with peaks detected. The y axis represents the read pileup normalized to the total number of reads at a base pair position (rpm/bp).

Article Snippet: Crosslinked chromatin (100 μg) extracted from NUGC-3 cells with mock and TNF-α (30 ng/mL) treatment was immunoprecipitated using 5 μg antibody against RELA (R&D Systems, catalog AF5078).

Techniques: Binding Assay