Journal: EMBO Reports

Article Title: The proximity-based protein interactome and regulatory logics of the transcription factor p65 NF-κB/RELA

doi: 10.1038/s44319-024-00339-8

Figure Lengend Snippet: ( A ) The left graph shows the X-ray crystal structure of a p50/p65 heterodimer bound to DNA as published in (Chen et al, ) (PDB 1kvx), while the right graph shows the entire p65 protein structure including the disordered C-terminal half as calculated by alphafold ( https://alphafold.ebi.ac.uk/entry/Q04206 ). Residues required for dimerization (Phe (F) 213, Leu (L) 215) or DNA binding (Glu (E) 39) are indicated in both structures. ( B ) Scheme of the HA-tagged p65-miniTurbo fusion proteins that were used to reconstitute p65-deficient HeLa cells under the control of a tetracycline-sensitive promoter. F213 and L215 in p65 wild type (wt) were mutated to Asp (FL/DD) for dimerization-deficient p65 or E39 to Ile (E/I) for DNA-binding-deficient p65. ( C ) Principle of proximity-based biotin tagging. ( D ) Pools of HeLa cells with CRISPR/Cas9-based suppression of endogenous p65/RELA (Δp65) were transiently transfected (using branched Polyethyleneimine, PEI) with the constructs shown in ( B ) and their expression was induced with doxycycline (1 µg/ml) for 17 h. At the end of this incubation, intracellular biotinylation was induced by adding 50 µM biotin for 70 min as indicated. Additionally, half of the samples were treated with IL-1α (10 ng/ml) for the last 60 min. Cell cultures expressing HA-miniTurbo only (empty vector, EV) or receiving only doxycycline or biotin served as negative controls (indicated by gray font). Parental HeLa cells (p) were included as further controls. Left panel: Cells were lysed and proteins were analyzed by Western blotting for the expression of p65-HA-miniTurbo and HA-miniTurbo using anti-p65 and anti-HA antibodies. Equal loading was confirmed by probing the blots with anti β-actin antibodies. Right panel: Biotinylated proteins from the same samples were purified on streptavidin-agarose beads and biotinylation patterns were visualized by Western blotting using streptavidin-horseradish peroxidase (HRP) conjugates (representative images from two independent experiments). ( E ) Biotinylated proteins from the experiment shown in ( C ) and from a second biological replicate were identified by mass spectrometry. Volcano plots show the ratio distributions of Log 2 -transformed mean protein intensity values on the X axes obtained with wild-type p65 or the p65 mutants compared to the empty vector controls in the presence or absence of IL-1α treatment. Y axes show corresponding p values from Student’s t test results. Strong enrichment of the bait p65/RELA proteins together with the core canonical NF-κB components is shown in red and blue colors, respectively (two biologically independent experiments and three technical replicates per sample). ( F ) Specific proteins binding to p65/RELA wild-type were defined by significant enrichment (Log 2 fold change (LFC) ≥ 2, −log 10 P ≥ 1.3, Student’s t test) compared to HA-miniTurbo only and to cells exposed to doxycycline or biotin only (see Fig. ). This set of proteins was intersected with proteins enriched in cells expressing p65 mutant proteins (LFC ≥ 2, −log10 P ≥ 1.3). Venn diagrams show the numbers of p65/RELA interactors and their overlaps before and after IL-1α-treatment, with values in the lower left corners indicating total numbers of interactors. ( G ) The six protein sets shown in ( E ) were subjected to parallel overrepresentation pathway analysis using Metascape software (Zhou et al, ). The Venn diagrams show the overlap of the top 100 enriched pathway terms. For IL-1α samples, only 92 terms were enriched. Values in the lower left corners indicate the total number of unique pathways. ( H ) The table shows the most strongly enriched pathway categories associated with the p65 /RELA wild-type or mutant interactomes. Numbers in brackets indicate the total numbers of p65/RELA interactors per condition according to ( E , F ). Enrichment P values for overrepresentation analyses were computed by Metascape software (Zhou et al, ). rtTA reverse tetracycline-controlled transactivator. The mass spectrometry data and bioinformatics analysis results are provided in Dataset . .

Article Snippet: Anti-NF-κB p65 (C-20) (rabbit polyclonal) for WB, ChIP, PLA , Santa Cruz Biotechnology , Cat.#sc-372 (discontinued).

Techniques: Binding Assay, Control, CRISPR, Transfection, Construct, Expressing, Incubation, Plasmid Preparation, Western Blot, Purification, Mass Spectrometry, Transformation Assay, Mutagenesis, Software

Journal: EMBO Reports

Article Title: The proximity-based protein interactome and regulatory logics of the transcription factor p65 NF-κB/RELA

doi: 10.1038/s44319-024-00339-8

Figure Lengend Snippet: ( A ) Biotinylated proteins from the experiments shown in Fig. and from a second biological replicate were identified by mass spectrometry in the presence or absence of IL-1α treatment of cells. Volcano plots show the ratio distributions of Log 2 transformed mean protein intensity values obtained with wild-type p65 in the presence of doxycycline and biotin (wt) compared to the empty vector control (EV) or compared with conditions in which only biotin (wt(bio)) or doxycycline (wt(dox)) were added to the cell cultures, to determine false positive values in the absence of expression of fusion protein but facilitated biotinylation, or in the absence of biotinylation but induced expression of the fusion protein, respectively. X axes show mean ratio value and Y axes show P values from t test results. Strong enrichment of the bait p65/RELA proteins together with the core canonical NF-κB components is shown in red and blue colors, respectively (two biologically independent experiments and three technical replicates per sample). ( B ) Specific proteins binding to p65/RELA wild type were defined by significant enrichment (LFC ≥ 2, −log 10 P ≥ 1.3, Student’s t test) compared to HA-miniTurbo only and to cells exposed to doxycycline or biotin only as shown in ( A ). Venn diagrams show the total numbers of specific p65/RELA interactors and their overlaps before and after IL-1α-treatment. The intersecting 279 (without IL-1α) and 310 (with IL-1α) interactors were pooled, resulting in the set of 366 specific p65/RELA interactors that was used for further downstream analyses. Numbers in the left lower corner of the boxes indicate the total number of detected interactors.

Article Snippet: Anti-NF-κB p65 (C-20) (rabbit polyclonal) for WB, ChIP, PLA , Santa Cruz Biotechnology , Cat.#sc-372 (discontinued).

Techniques: Mass Spectrometry, Transformation Assay, Plasmid Preparation, Control, Expressing, Binding Assay

Journal: EMBO Reports

Article Title: The proximity-based protein interactome and regulatory logics of the transcription factor p65 NF-κB/RELA

doi: 10.1038/s44319-024-00339-8

Figure Lengend Snippet: Pools of HeLa cells with CRISPR/Cas9-based suppression of endogenous p65/RELA (Δp65) were transiently transfected (using branched polyethyleneimine, PEI) with plasmids encoding HA-miniTurbo (empty vector, EV) or p65(wt) -HA-mTb or were left untransfected. Expression of HA-mTb or p65-HA-mTb(wt) was induced with doxycycline (1 µg/ml) for 17 h. Intracellular biotinylation was induced by the addition of 50 µM biotin for further 60 min during which time half of the samples were additionally treated with IL-1α (10 ng/ml). Cytosolic and nuclear fractions were prepared from cell lysates and proteins were analyzed by Western blotting for the subcellular expression of HA-miniTurbo, p65-HA-miniTurbo or the endogenous p50 NF-κB subunit using anti-HA, anti-p65 and anti-p50 antibodies, respectively. Antibodies against tubulin and P-Pol II were used to control separation of cell fractions. Equal loading of fractions was confirmed by probing the blots with anti β-actin antibodies. Shown is one representative out of two experiments.

Article Snippet: Anti-NF-κB p65 (C-20) (rabbit polyclonal) for WB, ChIP, PLA , Santa Cruz Biotechnology , Cat.#sc-372 (discontinued).

Techniques: CRISPR, Transfection, Plasmid Preparation, Expressing, Western Blot, Control

Journal: EMBO Reports

Article Title: The proximity-based protein interactome and regulatory logics of the transcription factor p65 NF-κB/RELA

doi: 10.1038/s44319-024-00339-8

Figure Lengend Snippet: ( A ) Venn diagram of p65/RELA interactors in IL-1α or untreated cells revealing the principal set of 366 unique p65/RELA interactors that were further investigated in this study. ( B ) Scheme showing the results of mapping the 366 interactors from ( A ) to the STRING database of protein-protein interactions (PPI) (Szklarczyk et al, ). ( C ) Scheme visualizing a set of 199 proteins enriched in the experimentally determined p65/RELA proximity interactome that have documented PPIs with the 46 known p65/RELA interactors in STRING. ( D ) Classification of the 366 p65/RELA interactors. ( E ) Protein interaction network of the 46 known p65/RELA interactors found by miniTurboID. Edge widths visualize the evidence for experimental interactions deposited in the STRING database. Nodes are colored in red and are arranged according to the enrichment found by proximity labeling in our study. .

Article Snippet: Anti-NF-κB p65 (C-20) (rabbit polyclonal) for WB, ChIP, PLA , Santa Cruz Biotechnology , Cat.#sc-372 (discontinued).

Techniques: Protein-Protein interactions, Labeling

Journal: EMBO Reports

Article Title: The proximity-based protein interactome and regulatory logics of the transcription factor p65 NF-κB/RELA

doi: 10.1038/s44319-024-00339-8

Figure Lengend Snippet: ( A ) Pools of HeLa cells with CRISPR/Cas9-based suppression of endogenous p65/RELA (Δp65) were transiently transfected (using branched polyethyleneimine, PEI) with plasmids encoding HA-miniTurbo (empty vector, EV) or p65(wt)-HA-mTb or were left untransfected. Expression of HA-mTb or p65(wt)-HA-mTb was induced with doxycycline (1 µg/ml) for 17 h. Intracellular biotinylation was induced by the addition of 50 µM biotin for further 60 min during which time half of the samples were additionally treated with IL-1α (10 ng/ml) for 30 or 60 min. Whole-cell extracts were prepared in urea buffer and (phospho-)proteins were analyzed by Western blotting for the expression of HA-miniTurbo or p65-HA-miniTurbo or the modification of p65-HA-mTb using the indicated antibodies. Equal loading was confirmed by probing the blots with anti β-actin antibodies. ( B ) Phospho-protein bands of p65-HA-mTb were normalized to the expression of p65-HA-mTb and changes were quantified relative to the corresponding untreated conditions of cells reconstituted with p65-HA-mTb. Bar graphs show data points and mean values ± s.d. from two or three independent experiments. The small increase of S468, S529 and S536 phosphorylation at 0.5 h of IL-1α-stimulation is not significant (according to one-way ANOVA). ( C ) The mass spectra of the p65-HA-mTb interactome analyses described in Fig. were re-investigated for phosphorylated peptides of p65/RELA. The table shows peptide sequences and the positions of amino acids with mass changes indicating phosphorylation. ( D ) The left graph shows (phospho-)peptide intensities for p65-HA-mTb across all conditions and the right graphs shows proportion of modified peptides.

Article Snippet: Anti-NF-κB p65 (C-20) (rabbit polyclonal) for WB, ChIP, PLA , Santa Cruz Biotechnology , Cat.#sc-372 (discontinued).

Techniques: CRISPR, Transfection, Plasmid Preparation, Expressing, Western Blot, Modification, Phospho-proteomics

Journal: EMBO Reports

Article Title: The proximity-based protein interactome and regulatory logics of the transcription factor p65 NF-κB/RELA

doi: 10.1038/s44319-024-00339-8

Figure Lengend Snippet: ( A ) The mass spectra of the 366 p65-HA-mTb interactors were re-investigated for phosphorylated amino acids resulting in 185 phospho-peptides representing 56 unique proteins. The table shows 14 phospho-peptides from 13 unique proteins with intensity values in all p65-HA-mTb samples and their regulation by IL-1α along with the HA-mTb negative controls. Phosphorylation sites are colored in red. ( B ) Immunoblots showing phosphorylation of c-JUN at Ser 63 in whole cells extracts of untreated or IL-1α-treated Δp65 cells transiently expressing p65-HA-mTb or HA-mTb as described in Fig. . ( C ) Volcano plots visualizing c-JUN and known c-JUN interactors (based on STRING entries) that were significantly enriched with p65(wt)-HA-mTb (LFC ≥ 2, −log10 P ≥ 1.3, Student’s t test) compared with HA-mTb (empty vector control, EV) before and after IL-1α treatment. ( D ) STRING-based protein interaction networks of c-JUN interactors according to their IL-1α-dependent enrichment in the biotinylated p65(wt)-HA-mTb interactome. Phosphorylated proteins (P) shown in ( A ) are indicated. ( E ) Similar networks were constructed for FOSL2, KMT2D and NCOR2 interactors. Known p65/RELA interactors (based on STRING) are colored in light blue.

Article Snippet: Anti-NF-κB p65 (C-20) (rabbit polyclonal) for WB, ChIP, PLA , Santa Cruz Biotechnology , Cat.#sc-372 (discontinued).

Techniques: Phospho-proteomics, Western Blot, Expressing, Plasmid Preparation, Control, Construct

Journal: EMBO Reports

Article Title: The proximity-based protein interactome and regulatory logics of the transcription factor p65 NF-κB/RELA

doi: 10.1038/s44319-024-00339-8

Figure Lengend Snippet: ( A ) The top-10 pathway terms according to GO (BP, CC, MF), KEGG, Reactome, STRING clusters and WikiPathways database entries and the top-10 subcellular localizations associated with the 366 p65/RELA interactors. Annotations, number of components and false discovery rates (FDR) were retrieved using the STRING plugin of Cytoscape (Shannon et al, ). ( B ) Overlap of the RELA interactome with 1639 human TFs (Lambert et al, ) and 801 epigenetic regulators (Marakulina et al, ). ( C ) Venn diagram showing the overlap of enriched TFs in basal or IL-1α-stimulated conditions. ( D ) Distribution of TF families found to be associated with p65/RELA in basal and IL-1α-stimulated conditions according to the annotation provided by (Lambert et al, ). ( E ) IL-1α-dependent enrichment of all TF belonging to ZBTB and ZNF families as identified by miniTurboID. ( F ) Volcano plots visualizing all TFs significantly enriched with wt p65/RELA (LFC ≥ 2, −log 10 P ≥ 1.3) compared with empty vector control (EV) and the changes obtained with p65 mutants in basal or IL-1α-stimulated conditions. ( G ) Graphs visualizing the top-10 enriched epigenetic regulators. Volcano plots show the ratio distributions of Log 2 transformed mean protein intensity values obtained with wild-type p65/RELA (wt) or with p65/RELA mutants (FL/DD, E/I) compared to empty vector controls (EV). Only 9 reader proteins were found. ( H ) Association of enriched epigenetic regulators with known epigenetic complexes according to the annotation provided by (Marakulina et al, ). Numbers in brackets show identified components per complex. ( E – G ) P values were calculated by Student’s t test. .

Article Snippet: Anti-NF-κB p65 (C-20) (rabbit polyclonal) for WB, ChIP, PLA , Santa Cruz Biotechnology , Cat.#sc-372 (discontinued).

Techniques: Plasmid Preparation, Control, Transformation Assay

Journal: EMBO Reports

Article Title: The proximity-based protein interactome and regulatory logics of the transcription factor p65 NF-κB/RELA

doi: 10.1038/s44319-024-00339-8

Figure Lengend Snippet: ( A ) Final list of top ranking high-confidence interactors of p65/RELA selected for further studies. The heatmap shows the Log 2 transformed mean protein intensity values from technical triplicates of the two biological independent miniTurboID experiments, the enrichment ratio values compared to the empty vector (HA-miniTurbo) control (EV) and the regulation by IL-1α. With the exception of N4BP3, all proteins were identified by at least two peptides. ( B ) Graph showing that the top 38 p65/RELA interactors are largely devoid of known protein interactions based on STRING entries. According to STRING, only two factors (CEBPD and FOSL1) interact with p65/RELA. Node borders visualize the main functional annotations. ( C ) HeLa cells were transiently transfected for 48 h with 20 nM of siRNAs mixtures for 38 HCI and p65/RELA, a siRNA targeting luciferase, transfection reagent alone or were left untreated. Half of the cells per plate were treated for 1 h with IL-1α (10 ng/ml) at the end of the incubation. cDNAs were transcribed in lysates and amplicons for three NF-κB target genes, two housekeeping genes and all 38 HCI p65/RELA interactors were pre-amplified by linear PCR and then quantified by qPCR. Based on Ct values, mRNA levels were quantified and normalized against GUSB . The effects of knockdowns were calculated separately for basal and IL-1α-inducible conditions against the luciferase siRNA. The heatmap shows hierarchically Kmeans clustered mean ratio values derived from three biologically independent siRNA screens. As a positive control, RELA knockdowns were performed in parallel. Green colors highlight p65/RELA interactors selected for further analysis. ( D ) The miniTurboID enrichment of six p65/RELA interactors (green colors) chosen from ( C ) is shown. P values were calculated by Student’s t test. Source data for the siRNA screen are provided in Dataset . .

Article Snippet: Anti-NF-κB p65 (C-20) (rabbit polyclonal) for WB, ChIP, PLA , Santa Cruz Biotechnology , Cat.#sc-372 (discontinued).

Techniques: Transformation Assay, Plasmid Preparation, Control, Functional Assay, Transfection, Luciferase, Incubation, Amplification, Derivative Assay, Positive Control

Journal: EMBO Reports

Article Title: The proximity-based protein interactome and regulatory logics of the transcription factor p65 NF-κB/RELA

doi: 10.1038/s44319-024-00339-8

Figure Lengend Snippet: Proximity ligation assays coupled to immunofluorescence (IF) were performed with HeLa cells or Δp65 HeLa cells lacking endogenous p65/RELA to demonstrate interactions of p65/RELA with TFE3 ( A ), TFEB ( B ), GLIS2 ( C ) and ZBTB5 ( D ) using pairs of antibodies along with negative control conditions as indicated. PLA spots are colored in red, while p65 IF is colored in green. Nuclear DNA is counterstained with Hoechst 33342 (blue signals). The images show representative fluorescence raw data. The violin plots on the right show quantification of PLA spots per cell from the numbers of cells indicated in brackets as obtained from three (TFE3, TFEB, GLIS2) or two (ZBTB5) independent experiments. Samples lacking one or both primary antibodies (PLA ctr) served as negative controls. Experiments shown in ( A – C ) were performed in parallel with one set of PLA ctr and p65 gt antibody only samples that were included in each of the graphs shown in ( A – C ) for comparison. Solid lines indicate medians and dashed lines indicate 1st and 3rd quartiles. Asterisks indicate results from Kruskal-Wallis tests compared to the parental control (**** P ≤ 0.0001) obtained by one-way ANOVA. Scale bars indicate 50 µm. gt goat, ms mouse, rb rabbit.

Article Snippet: Anti-NF-κB p65 (C-20) (rabbit polyclonal) for WB, ChIP, PLA , Santa Cruz Biotechnology , Cat.#sc-372 (discontinued).

Techniques: Ligation, Immunofluorescence, Negative Control, Fluorescence, Comparison, Control

Journal: EMBO Reports

Article Title: The proximity-based protein interactome and regulatory logics of the transcription factor p65 NF-κB/RELA

doi: 10.1038/s44319-024-00339-8

Figure Lengend Snippet: ( A ) Volcano plots revealing the basal and IL-1α-dependent enrichment of all MiT/TFE and GLIS family members in the miniTurboID experiments. P values were calculated by Student’s t test. For details, see Fig. . ( B ) The subcellular distribution of phosphorylated (P) and dephosphorylated forms of TFE3 and TFEB, p50, and p65 NF-κB was evaluated by Western blotting in cell extracts from HeLa cells stimulated with IL-1α or subjected to starvation (HBSS) for the indicated times. “W” indicates samples washed four times with HBSS and then supplemented with their previous cell culture medium to control for effects caused by the washing procedure prior to addition of starvation medium. Antibodies against RNA polymerase (pol II), tubulin or β-actin served as control for fractionation and equal protein loading. Shown is one out of three biologically independent experiments. See Appendix Fig. for the quantification of replicates. ( C ) Parental HeLa cells or cells transfected with siRNAs (20 nM) against TFs or luciferase (as a negative control) were cultivated for 48 h. Then, half of the cells were stimulated for 1 h with IL-1α (10 ng/ml) or were left untreated. Total cell extracts were examined for the expression of the indicated proteins by Western blotting. Antibodies against β-actin served as loading controls. Shown is one out of three biologically independent experiments. ( D ) Quantification of the basal expression levels of the indicated TFs in extracts of cells transfected as in ( C ). Bar graphs show data points and mean values ± s.d. relative to parental cells from six biologically independent experiments. Asterisks indicate P values (* P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001) obtained by one-way ANOVA. ( E ) Total RNA isolated from cells treated as in ( C ) was analyzed for mRNA expression of the indicated NF-κB target genes by RT-qPCR. Bar graphs show data points and mean values ± s.d. relative to cells transfected with luciferase siRNA from three biologically independent experiments. siTFE3/B indicates double knockdown of TFE3 and TFEB. Asterisks indicate P values (* P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001) obtained by one-way ANOVA. .

Article Snippet: Anti-NF-κB p65 (C-20) (rabbit polyclonal) for WB, ChIP, PLA , Santa Cruz Biotechnology , Cat.#sc-372 (discontinued).

Techniques: Western Blot, Cell Culture, Control, Fractionation, Transfection, Luciferase, Negative Control, Expressing, Isolation, Quantitative RT-PCR, Knockdown

Journal: EMBO Reports

Article Title: The proximity-based protein interactome and regulatory logics of the transcription factor p65 NF-κB/RELA

doi: 10.1038/s44319-024-00339-8

Figure Lengend Snippet: ( A ) Schematic illustrating the strategy to analyze the influences of novel p65/RELA interactors on IL-1α-regulated p65/RELA target genes by combining siRNA-mediated knockdown with transcriptome analysis. ( B ) HeLa cells were transiently transfected for 48 h with 20 nM siRNA mixtures against RELA, ZBTB5, S100A8, S100A9 (series 1) or RELA, GLIS2, TFE3, TFEB (series 2) and an siRNA against luciferase (siLuc) as control. Half of the cells were treated with IL-1α (10 ng/ml) for 1 h at the end of incubation, and Agilent microarray analyses were performed from total RNA. Normalized data were used to identify DEGs based on an LFC ≥ 1 with a −log 10 P value ≥ 1.3 (moderated t test). Venn diagrams show the overlap of all DEGs that were affected at least twofold by siRNA knockdown in IL-1α-treated samples, with the ratio of siLuc to individual knockdown determined in each case. Red colors mark genes jointly regulated by knockdown of RELA and one of its interactors (two biologically independent experiments). ( C ) Violin plots show the distribution, medians, and interquartile ranges of normalized expression levels for all IL-1α-regulated genes and the corresponding changes in the gene subsets defined in Fig. 6B that were affected by siRNA knockdown. The number of these genes is indicated in parentheses. Asterisks indicate significant changes as determined by a two-tailed Mann–Whitney test (* P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001). ( D ) Superimposed pairwise correlation analyses of the mean ratio changes of all genes (gray), IL-1α-regulated genes (blue), and gene sets significantly up- or downregulated by siRNA knockdown (red). Ratio values from RELA knockdown conditions were compared with the knockdown of a RELA interactor in each case. Genes that are jointly regulated by knockdown of RELA and one of its interactors correspond to the Venn diagrams of ( B ) and are marked in red. Coefficients of correlation (Pearson’s r ), corresponding P values and coefficients of determination ( r 2 ) rare indicated for all comparisons. The complete set of data is provided in Dataset . .

Article Snippet: Anti-NF-κB p65 (C-20) (rabbit polyclonal) for WB, ChIP, PLA , Santa Cruz Biotechnology , Cat.#sc-372 (discontinued).

Techniques: Knockdown, Transfection, Luciferase, Control, Incubation, Microarray, Expressing, Two Tailed Test, MANN-WHITNEY

Journal: EMBO Reports

Article Title: The proximity-based protein interactome and regulatory logics of the transcription factor p65 NF-κB/RELA

doi: 10.1038/s44319-024-00339-8

Figure Lengend Snippet: ( A ) Schematic illustrating the strategy to project the protein interactions of all target genes defined by knockdowns of p65/RELA or its interactors in IL-1α-stimulated cells into combined functional networks. ( B ) Table summarizing the numbers of mapped IDs ( = nodes) corresponding to the gene groups shown in Fig. , their protein interactions ( = edges) and the protein interaction network enrichment P values as derived from algorithms embedded in STRING (Franceschini et al, ). ( C ) Cytoscape-derived PPI networks. Nodes are colored and arranged according to the deregulation of the corresponding genes by knockdown of p65/RELA or its interactors. Edges visualize known protein interactions, including the small number of interactions reported for p65/RELA, S100A8/9, and TFE3/TFEB. No interactions were found for ZBTB5 and GLIS2. .

Article Snippet: Anti-NF-κB p65 (C-20) (rabbit polyclonal) for WB, ChIP, PLA , Santa Cruz Biotechnology , Cat.#sc-372 (discontinued).

Techniques: Functional Assay, Derivative Assay, Knockdown

Journal: EMBO Reports

Article Title: The proximity-based protein interactome and regulatory logics of the transcription factor p65 NF-κB/RELA

doi: 10.1038/s44319-024-00339-8

Figure Lengend Snippet: ( A ) Schematic illustrating the strategy to use p65/RELA ChIPseq data for delineating chromatin recruitment of RELA together with its interactors on the basis of DNA motifs and three possible scenarios of interactions. ( B ) Windows of 1000 base pairs surrounding experimentally determined p65/RELA ChIPseq peaks (Data ref: Jurida et al, ; Jurida et al, ) were searched for motifs of RELA and REL TFs using matrices from the JASPAR database. P values indicated significant enrichment compared to the whole genome calculated by MEME-ChIP (Ma et al, ). The Venn diagram shows the overlap and inserts show motif compositions. ( C ) Venn diagrams indicating the overlap of motifs found for RELA or the RELA interactors TFE3, TFEB or GLIS2 in chromosomal regions assigned to p65/RELA ChIPseq peaks. P values indicate significant enrichment compared to the whole genome calculated by MEME-ChIP (Ma et al, ). Inserts show motif compositions. ( D ) All target genes that were significantly up- or downregulated under basal or IL-1α-stimulated conditions as shown in Fig. or Appendix Fig. were collected and were examined for their association with a p65/RELA ChIPseq peak. The pie charts show the numbers of RELA, TFE3, TFEB and GLIS2 motifs detected in siRNA RELA target genes with an annotated p65/RELA peak in their promoters or enhancers. ( E ) Overlap of all genes with p65/RELA peaks in promoters or enhancers and at least one motif for the indicated transcription factors in IL-1α-stimulated conditions. ( F ) Genome browser view of the TNFAIP3 locus with p65/RELA ChIPseq peaks, activated enhancers and promoters (H3K27ac), accessible chromatin (ATACseq) and mRNA production (RNAseq) before and after 1 h of IL-1α stimulation. Data sets were from GSE64224, GSE52470 and GSE134436 and are aligned to HG19 (Data ref: Handschick et al, ; Data ref: Weiterer et al, ; Data ref:Jurida et al, ; Handschick et al, ; Jurida et al, ; Weiterer et al, ). p65/RELA binding regions of 1000 bp under p65/RELA peaks and identified TF motifs are indicated by horizontal lines. ( G ) KB cells were left untreated or were starved for 24 h in HBSS. Half of the cells was treated with IL-1α (10 ng/ml) for 1 h before the end of the experiment. ChIP-qPCR was performed with the indicated antibodies or IgG controls and a primer pair covering the TNFAIP3 promoter region (marked with an arrow in Fig. 8F) and relative enrichment of TNFAIP3 promoter fragments was calculated (percent input). Box plots show all data points with means and minimum/maximum values from three independent biological replicates performed with two technical replicates. The complete set of data is provided in Dataset . .

Article Snippet: Anti-NF-κB p65 (C-20) (rabbit polyclonal) for WB, ChIP, PLA , Santa Cruz Biotechnology , Cat.#sc-372 (discontinued).

Techniques: Binding Assay, ChIP-qPCR

Journal: EMBO Reports

Article Title: The proximity-based protein interactome and regulatory logics of the transcription factor p65 NF-κB/RELA

doi: 10.1038/s44319-024-00339-8

Figure Lengend Snippet: Reagents and tools table

Article Snippet: Anti-NF-κB p65 (C-20) (rabbit polyclonal) for WB, ChIP, PLA , Santa Cruz Biotechnology , Cat.#sc-372 (discontinued).

Techniques: CRISPR, Bacteria, Recombinant, Cloning, Clone Assay, Mutagenesis, Control, Plasmid Preparation, Sequencing, Luciferase, Gene Expression, Labeling, Binding Assay, Modification, Saline, Western Blot, Transfection, Protease Inhibitor, Random Hexamer, Reverse Transcription, Membrane, In Situ, Proximity Ligation Assay, SYBR Green Assay, Microarray, Software

Journal: International Journal of Molecular Sciences

Article Title: Effects of Fasting on THP1 Macrophage Metabolism and Inflammatory Profile

doi: 10.3390/ijms25169029

Figure Lengend Snippet: SD effects on NF-κB activation. Immunofluorescence of p65 subunit of NF-κB in macrophages under the FCS conditions described in . In control and SD groups, p65 subunit of NFkB remains in cytoplasmatic localization. Nuclear translocation was mainly observed in ISD groups ( n = 3).

Article Snippet: The staining was performed by incubating with NFκB p65 antibody (C-20) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and secondary antibody goat anti-rabbit AlexaFluor-488 (A11008, Thermofisher) previous to mounting with aqueous medium.

Techniques: Activation Assay, Immunofluorescence, Control, Translocation Assay