pe-labelled crel antibody  (Thermo Fisher)

Journal: RSC Medicinal Chemistry

Article Title: Design, synthesis and evaluation of pyrrolobenzodiazepine (PBD)-based PROTAC conjugates for the selective degradation of the NF-κB RelA/p65 subunit †

doi: 10.1039/d5md00316d

Figure Lengend Snippet: Comparison the effect of 15d (PROTAC) on the expression of three NF-κB subunits RelA/p65, RelB and cRel. Each subunit was quantified using fluorescence-labelled antibodies; all experiments were performed three times in duplicate and data are presented as violin plots. Statistical significance was determined using the Kruskal–Wallis test with Dunn's multiple comparison post hoc correction. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Cells were then washed in Cyto-FastTM Perm Wash solution and centrifuged at 300 × g for 5 min before being resuspended in 100 μL Perm Wash solution followed by the addition of 5 μL APC-labelled RelA/p65 antibody (Biolegend), 5 μL PE-labelled cRel antibody (eBiosciences) and 5 μL corallite 488-labelled RelB antibody (ThermoFisher).

Techniques: Comparison, Expressing, Fluorescence

Journal: bioRxiv

Article Title: NF-κB is a Central Regulator of Hypoxia-Induced Gene Expression

doi: 10.1101/2025.01.08.631917

Figure Lengend Snippet: A. RNA-seq (n=2) in HeLa cells cultured at 21% oxygen (normoxia) or exposed to 24 h 1% oxygen (hypoxia), transfected with control siRNA or RelA, RelB or cRel siRNAs. B-E. Differential expression analysis volcano plots for 24 h hypoxia control, siRelA, siRelB or sicRel compared to normoxia control. Blue points indicate DEGs. F-G. Overlap of siRelA, siRelB and sicRel upregulated genes in hypoxia, compared to normoxia control ( F ) and siRelA, siRelB and sicRel downregulated genes in hypoxia, compared to normoxia control ( G ) in HeLa cells.

Article Snippet: The following primary antibodies were used for immunoblotting: RelA (sc-8008, Santa Cruz), RelB (10544, Cell Signalling), cRel (4727, Cell Signalling), HIF-1α (610958, BD Biosciences), β-Actin (66009-1-lg, Proteintech), IDH1 (12332-1-AP, Proteintech), SOD1 (sc-17767, Santa Cruz), Total Oxphos Antibody Cocktail for detecting ATP5A, UQCRC2, and SDHB (ab110411, abcam).

Techniques: RNA Sequencing Assay, Cell Culture, Transfection, Control, Expressing

Journal: bioRxiv

Article Title: NF-κB is a Central Regulator of Hypoxia-Induced Gene Expression

doi: 10.1101/2025.01.08.631917

Figure Lengend Snippet: A-B. NF-κB dependence of hypoxia inducible Differential Expressed Genes (DEGs); percentage of NF-κB dependent DEGs are labelled. C. Heatmap displaying Z score transformed gene expression levels for NF-κB dependent hypoxia regulated genes. D-E. Overlap of RelA, RelB or cRel-dependent hypoxia up- or down-regulated DEGs. F-G. Over Representation Analysis (ORA) was performed through WEB-based Gene SeT AnaLysis Toolkit using the Molecular Signatures Database hallmark gene sets, investigating NF-κB dependent hypoxia upregulated DEGs ( F ) or downregulated DEGs ( G ). Dashed line shows statistical significance threshold of FDR 0.05 (-Log 10 FDR 1.3). ( H ) Overlap of NF-κB dependent hypoxia up- and down-regulated DEGs identified by RNA-seq (n=2) with NF-κB subunit binding sites acquired from ChIP-seq Atlas database. Percentage of DEGs containing an NF-κB subunit binding site are displayed; statistical significance of the gene list overlaps was determined via hypergeometric test, *** P < 0.001.

Article Snippet: The following primary antibodies were used for immunoblotting: RelA (sc-8008, Santa Cruz), RelB (10544, Cell Signalling), cRel (4727, Cell Signalling), HIF-1α (610958, BD Biosciences), β-Actin (66009-1-lg, Proteintech), IDH1 (12332-1-AP, Proteintech), SOD1 (sc-17767, Santa Cruz), Total Oxphos Antibody Cocktail for detecting ATP5A, UQCRC2, and SDHB (ab110411, abcam).

Techniques: Transformation Assay, Expressing, RNA Sequencing Assay, Binding Assay, ChIP-sequencing

Journal: bioRxiv

Article Title: NF-κB is a Central Regulator of Hypoxia-Induced Gene Expression

doi: 10.1101/2025.01.08.631917

Figure Lengend Snippet: A. Log 2 Fold change of selected NF-κB dependent hypoxia up- and down-regulated DEGs showing altered transcript levels in hypoxia compared to normoxia identified by RNA-seq (n=2). B-F. qPCR analysis in HeLa cells cultured at 21% oxygen (control) or 24 h 1% oxygen (hypoxia), transfected with control siRNA or RelA, RelB or cRel siRNAs. RelA- ( B ), RelB- ( C ), and cRel-dependent ( D ) hypoxia upregulated DEGs. RelA- ( E ) and RelB-dependent ( F ) hypoxia downregulated DEGs. Relative mRNA expression levels of the indicated genes were analysed using 18S as a normalising gene. Graphs show mean (n=4) ± SEM, * P < 0.05, ** P < 0.01, *** P < 0.001. Statistical significance was determined via one-way ANOVA with post-hoc Dunnett’s test.

Article Snippet: The following primary antibodies were used for immunoblotting: RelA (sc-8008, Santa Cruz), RelB (10544, Cell Signalling), cRel (4727, Cell Signalling), HIF-1α (610958, BD Biosciences), β-Actin (66009-1-lg, Proteintech), IDH1 (12332-1-AP, Proteintech), SOD1 (sc-17767, Santa Cruz), Total Oxphos Antibody Cocktail for detecting ATP5A, UQCRC2, and SDHB (ab110411, abcam).

Techniques: RNA Sequencing Assay, Cell Culture, Control, Transfection, Expressing

Journal: bioRxiv

Article Title: NF-κB is a Central Regulator of Hypoxia-Induced Gene Expression

doi: 10.1101/2025.01.08.631917

Figure Lengend Snippet: A. Cellular ROS measurements using CellRox staining and immunofluorescence analysis in HeLa cells, cultured at 21% oxygen (control) or 24 h 1% oxygen (hypoxia), transfected with control siRNA or RelA, RelB or cRel siRNAs. Representative images from 3 independent experiments are shown. B. Quantification of relative ROS signal in individual cells, mean (n=3) ± SEM, * P < 0.05, ** P < 0.01, *** P < 0.001. Statistical significance was determined via one-way ANOVA with post-hoc Dunnett’s test. C. Immunoblot analysis of the indicated protein in HeLa cells exposed or not to 1% oxygen for 24 h, with siRNA transfection of control, RelA, RelB or cRel. D. Signal intensity normalised to actin (mean n=3, ± SEM) from immunoblot analysis in ( C ). * P < 0.05, ** P < 0.01, *** P < 0.001. Statistical significance was determined via one-way ANOVA with post-hoc Dunnett’s test.

Article Snippet: The following primary antibodies were used for immunoblotting: RelA (sc-8008, Santa Cruz), RelB (10544, Cell Signalling), cRel (4727, Cell Signalling), HIF-1α (610958, BD Biosciences), β-Actin (66009-1-lg, Proteintech), IDH1 (12332-1-AP, Proteintech), SOD1 (sc-17767, Santa Cruz), Total Oxphos Antibody Cocktail for detecting ATP5A, UQCRC2, and SDHB (ab110411, abcam).

Techniques: Staining, Immunofluorescence, Cell Culture, Control, Transfection, Western Blot

Journal: bioRxiv

Article Title: NF-κB is a Central Regulator of Hypoxia-Induced Gene Expression

doi: 10.1101/2025.01.08.631917

Figure Lengend Snippet: A. Immunoblot analysis of the indicated proteins in HeLa cells cultured at 21% oxygen (control) or 24 h 1% oxygen (hypoxia), transfected with control siRNA or RelA, RelB or cRel siRNAs. Representative images from 3 independent experiments are shown. B. Signal intensity normalised to actin (mean n=3, ± SEM) from immunoblot analysis in ( A ). * P < 0.05, ** P < 0.01, *** P < 0.001. Statistical significance was determined via one-way ANOVA with post-hoc Dunnett’s test.

Article Snippet: The following primary antibodies were used for immunoblotting: RelA (sc-8008, Santa Cruz), RelB (10544, Cell Signalling), cRel (4727, Cell Signalling), HIF-1α (610958, BD Biosciences), β-Actin (66009-1-lg, Proteintech), IDH1 (12332-1-AP, Proteintech), SOD1 (sc-17767, Santa Cruz), Total Oxphos Antibody Cocktail for detecting ATP5A, UQCRC2, and SDHB (ab110411, abcam).

Techniques: Western Blot, Cell Culture, Control, Transfection

Journal: bioRxiv

Article Title: Systems biology-enabled targeting of NF-κB and BCL2 overcomes microenvironment-mediated BH3-mimetic resistance in DLBCL

doi: 10.1101/2024.11.30.626166

Figure Lengend Snippet: Immunofluorescence microscopy reveals differential NF-κB basal RelA activity in Diffuse Large B-cell Lymphoma (DLBCL) cell lines. (A) Schematic of the canonical and non-canonical NF-κB signaling pathway illustrating how non-canonical NF-κB activation results in the induction of BCLXL and how elevated basal canonical signaling induces multiple negative regulators of NF-κB which may contribute to cell line specific induction of MCL1. (B) Representative immunofluorescence microscopy following staining in RIVA cells for RelA (NF-κB), DAPI (nucleus), actin (cytoplasm), to measure nuclear and cytoplasmic subcellular localization. (C) Quantification immunofluorescence microscopy showing nuclear to cytoplasmic ratio of cRel and RelA in single cells. Data shows a representative replicate in the indicated cell lines. Violin width indicates data density. (D) Quantification of the nuclear to cytoplasmic ratio of cRel (top) and RelA (bottom) as bar graphs with error bars of the mean ± standard deviation of three independent experiments in RIVA, U2932 and SUDHL8 (*P<0.1; One-Way ANOVA).

Article Snippet: Cells were stained with anti-p65 (Invitrogen), RelB (Cell signaling), cRel (Invitrogen), DAPI (ThermoFisher) and rhodamine phalloidin (Thermo Scientific).

Techniques: Immunofluorescence, Microscopy, Activity Assay, Activation Assay, Staining, Standard Deviation

Journal: bioRxiv

Article Title: Systems biology-enabled targeting of NF-κB and BCL2 overcomes microenvironment-mediated BH3-mimetic resistance in DLBCL

doi: 10.1101/2024.11.30.626166

Figure Lengend Snippet: Co-culture with hCD40L-3T3 cells upregulates NF-κB RelB and BCLXL in DLBCL, and selectively upregulates NF-κB cRel and MCL1 through signaling crosstalk when NF-κB RelA is chronically active. (A) Abundances of the heterodimers RelA:p50 (left) and cRel:p50 (right) bound to inhibiting NF-κB proteins (IκB), as simulated by computational modeling for basal canonical NF-κB activation state in RIVA, and high basal activation state in SUDHL8. (B) Computational modeling results showing the nuclear abundance of the indicated NF-κB dimers in a simulation with low basal nuclear RelA (RIVA, pink), and a simulation high basal nuclear RelA (SUDHL8, teal). The mean (line) and standard deviation (shaded region) of 25 cells is indicated. (C) Schematic demonstrating the mechanism by which NF-κB contributes to the induction of BCLXL in a cell line with normal basal signaling (RIVA) upon stimulation with CD40L (right). hCD40L-3T3 mediated activation of NIK results in p100 processing into p52, leading to nuclear translocation of RelB:p52 and increased expression of BCLXL. Gray = inactive pathways and low abundance proteins, color = active pathways and predominant complexes. (D) Schematic demonstrating how crosstalk emerges between CD40, NIK, cRel, and MCL1. Increase basal RelA results in increase p100, and IκBδ (left). hCD40L-3T3 mediated activation of NIK results in processing of IκBδ and release of cRel:p50, which translocates to the nucleus and potentially upregulates expression of MCL1 (right). Gray = inactive pathways and low abundance proteins, color = active pathways and predominant complexes. (E-F) Quantification of immunofluorescence microscopy in RIVA (E) and SUDHL8 (F) cells showing nuclear to cytoplasmic ratio of RelA, RelB and cRel. Data post 24 hours of monoculture and hCD40L-3T3co-culture is shown side by side. Single cells of a representative replicate are shown (left), with violin width indicating data density. Quantification of the nuclear to cytoplasmic ratio of RelA, RelB and cRel is shown as bar graphs (right) with error bars displaying the mean ± standard deviation of three independent experiments ( *P<0.5, **P<0.01 ; unpaired t-test). (G) Schematic of the co-culture system used, following 24 hours of treatment with 0.0001-100μΜ of the BCLXL inhibitor A1331852 ± 0.1μM of the Bruton’s Kinase (BTK) inhibitor Ibrutinib. (H) Cell viability of SUDHL8 in response to 0.0001-100μΜ of the BCLXL inhibitor A1331852 post a 24-hour treatment in monoculture, post a 24-hour hCD40L-3T3co-culture or post a 24-hour hCD40L-3T3 co-culture with the addition of 0.1μM of the BTK inhibitor, Ibrutinib. LC 50 values for each condition is shown (right) with error bars representing the mean ± standard deviation of three independent experiments, normalized to the untreated control ( ***P<0.001 one-way ANOVA with Tukey’s comparisons test).

Article Snippet: Cells were stained with anti-p65 (Invitrogen), RelB (Cell signaling), cRel (Invitrogen), DAPI (ThermoFisher) and rhodamine phalloidin (Thermo Scientific).

Techniques: Co-Culture Assay, Activation Assay, Standard Deviation, Translocation Assay, Expressing, Immunofluorescence, Microscopy, Control

Journal: bioRxiv

Article Title: Systems biology-enabled targeting of NF-κB and BCL2 overcomes microenvironment-mediated BH3-mimetic resistance in DLBCL

doi: 10.1101/2024.11.30.626166

Figure Lengend Snippet: IκBε knock-out mouse model reveals cRel-dependent induction of MCL1. (A) NF-κB ChIP-seq data from Zhao et al. 2014 showing binding of the indicated NF-κB subunits around the transcription start site (TSS) of the indicated anti-apoptotic genes in lymphoblastoid B cell line. A 1kb window around the TSS is indicated. (B) Model-based Analysis for ChIP-Seq 2 (MACS2) scores for the indicated NF-κB subunits within 1kb of the MCL1 promoter across all ChIP-Seq datasets reported in ChIP Atlas Zou et al. 2024 , after outlier removal using ROUT method (q=1%, ****p<0.0001). (C) Schematic of normal basal NF-κB signaling (left), schematic of NF-κB signaling predicting the unknown effect of IκBε −/ - on cRel and MCL-1 induction. (D) Computational modelling simulation of RelA and cRel abundance in IκBε −/ - and IκBα κB/κB demonstrated as fold changes normalized to wild-type. Error bars represent the mean and ± standard deviation of 25 cells (***P<0.001 , unpaired t-test). (E) RelA and cRel abundances in IκBε −/− and IκBα κB/κB cells derived from primary splenocytes, demonstrated as fold changes to wild-type, error bars representing the mean ± standard deviation. (F) Experimental pipeline showing the workflow of isolating and purifying primary B cells from wild type, IκBε − / − and IκBα κB/κB mouse genotypes. (G) Abundances of MCL1 and BCLXL acquired with flow cytometry in primary B cells isolated and purified from IκBε − / − (left) and IκBα κB/κB (right) mouse genotypes, demonstrated as fold changes normalized to wild-type. Error bars represent the mean ± standard deviation of three independent experiments ( *P<0.05 , unpaired t-test).

Article Snippet: Cells were stained with anti-p65 (Invitrogen), RelB (Cell signaling), cRel (Invitrogen), DAPI (ThermoFisher) and rhodamine phalloidin (Thermo Scientific).

Techniques: Knock-Out, ChIP-sequencing, Binding Assay, Standard Deviation, Derivative Assay, Flow Cytometry, Isolation, Purification

Journal: Cell

Article Title: Thyroid hormone remodels cortex to coordinate body-wide metabolism and exploration

doi: 10.1016/j.cell.2024.07.041

Figure Lengend Snippet:

Article Snippet: DRH032_ AAV-SIO-nEF-DN-THR , This paper , Addgene 225091.

Techniques: Virus, Plasmid Preparation, Recombinant, Control, Protease Inhibitor, RNAscope, Multiplex Assay, Negative Control, Software, Modification

Journal: Cell

Article Title: Thyroid hormone remodels cortex to coordinate body-wide metabolism and exploration

doi: 10.1016/j.cell.2024.07.041

Figure Lengend Snippet:

Article Snippet: DRH031_ AAV-SIO-nEF-WT-THR , This paper , Addgene 225090.

Techniques: Virus, Plasmid Preparation, Recombinant, Control, Protease Inhibitor, RNAscope, Multiplex Assay, Negative Control, Software, Modification