nf κb1 p105 p50 d7h5m  (Cell Signaling Technology Inc)

Journal: JCI Insight

Article Title: Heterozygous NFKB1 variant causes inflammatory dysregulation shaped by broader genetic context in common variable immunodeficiency

doi: 10.1172/jci.insight.198703

Figure Lengend Snippet: ( A ) Difficulty to maintain healthy weight persists despite several therapeutic approaches. TPN, total peripheral nutrition. ( B ) Family pedigree for heterozygous 1377delT NFKB1 variant. ( C ) Schematic illustration of NFKB1 , p50, and p105 coding regions as denoted. Location of 1377delT variant as indicated in red. RHD, Rel homology domain; ARD, ankyrin repeat domain; DD, death domain. ( D ) NFKB1 sequence from PBMCs by Sanger sequencing for healthy donor (HD) on left and CA01 on right. 1377delT frameshift denoted in red.

Article Snippet: The primary antibodies used were p105/p50 (Cell Signaling Tech, 3035S), RelB (Cell Signaling Tech, 4954S), C-Rel (Cell Signaling Tech, 4727S), p100/p52 (Cell Signaling Tech, 4882S), and p65 (Cell Signaling Tech, 3035S).

Techniques: Variant Assay, Sequencing

Journal: JCI Insight

Article Title: Heterozygous NFKB1 variant causes inflammatory dysregulation shaped by broader genetic context in common variable immunodeficiency

doi: 10.1172/jci.insight.198703

Figure Lengend Snippet: ( A ) Schematic of protocol used to generate monocytes from iPSCs. ( B ) Morphology and DNA sequence in region of 1377delT NFKB1 variant (highlighted in yellow) for each iPSC line used in this study. ( C ) Flow cytometry of CD45/CD14 and CD16/CD163 on iMONOs generated from each iPSC line used in this study. Data representative of more than 12 iMONO differentiations. Genetic background of iPSC line and presence of heterozygous NFKB1 variant as denoted.

Article Snippet: The primary antibodies used were p105/p50 (Cell Signaling Tech, 3035S), RelB (Cell Signaling Tech, 4954S), C-Rel (Cell Signaling Tech, 4727S), p100/p52 (Cell Signaling Tech, 4882S), and p65 (Cell Signaling Tech, 3035S).

Techniques: Sequencing, Variant Assay, Flow Cytometry, Generated

Journal: JCI Insight

Article Title: Heterozygous NFKB1 variant causes inflammatory dysregulation shaped by broader genetic context in common variable immunodeficiency

doi: 10.1172/jci.insight.198703

Figure Lengend Snippet: ( A ) Schematic of protocol for iMONO assay. ( B ) Whole cell iMONO protein lysate (20 μg) analyzed for p105/p50 protein level by Western blot. Source of iPSC and present of NFKB1 variant as indicated. ( C ) Densitometry analysis of Western blot bands from 3 blots generated from 3 independent experiments. P value was calculated by 1-way ANOVA with Holm-Šídák test for multiple comparisons. * P < 0.05.

Article Snippet: The primary antibodies used were p105/p50 (Cell Signaling Tech, 3035S), RelB (Cell Signaling Tech, 4954S), C-Rel (Cell Signaling Tech, 4727S), p100/p52 (Cell Signaling Tech, 4882S), and p65 (Cell Signaling Tech, 3035S).

Techniques: Western Blot, Variant Assay, Generated

Journal: JCI Insight

Article Title: Heterozygous NFKB1 variant causes inflammatory dysregulation shaped by broader genetic context in common variable immunodeficiency

doi: 10.1172/jci.insight.198703

Figure Lengend Snippet: ( A ) IL-1β, CXCL1, and CXCL2 measured in supernatant of iMONO cultures with and without LPS. ( B ) IL-12, CCL4, and CCL22 measured in supernatant of iMONO cultures with and without LPS. Parental iPSC line (CA01 or HD) and presence of WT or 1377delT NFKB1 for both alleles as noted. Each data point represents an experimental replicate. P value was calculated by 2-way ANOVA with Holm-Šídák test for multiple comparisons. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: The primary antibodies used were p105/p50 (Cell Signaling Tech, 3035S), RelB (Cell Signaling Tech, 4954S), C-Rel (Cell Signaling Tech, 4727S), p100/p52 (Cell Signaling Tech, 4882S), and p65 (Cell Signaling Tech, 3035S).

Techniques:

Journal: JCI Insight

Article Title: Heterozygous NFKB1 variant causes inflammatory dysregulation shaped by broader genetic context in common variable immunodeficiency

doi: 10.1172/jci.insight.198703

Figure Lengend Snippet: ( A ) PCA plot of RNA-seq from iMONO cultures for CA01 and HD with and without heterozygous 1377delT NFKB1 variant. ( B and C ) Volcano plots illustrating distribution of RNA-seq changes from iMONO cultures with and without heterozygous 1377delT NFKB1 variant in CA01 ( B ) and HD backgrounds ( C ). ( D ) Top 10 affected pathways by P value from HALLMARK pathway clustergrams of gene upregulated by heterozygous 1377delT NFKB1 variant in iMONOs from CA01 and HD genetic backgrounds 0, 4, and 6 hours culture with LPS. EMT, epithelial mesenchymal transition. ( E ) Gene expression increased by 1377delT NFKB1 in CA01, but not HD, iMONOs. ( F ) Genes from TNF signaling and inflammatory response gene sets with increased expression due to heterozygous 1377delT NFKB1 in CA01, but not HD, iMONOs. Significant pathways in red. PCA, principal component analysis.

Article Snippet: The primary antibodies used were p105/p50 (Cell Signaling Tech, 3035S), RelB (Cell Signaling Tech, 4954S), C-Rel (Cell Signaling Tech, 4727S), p100/p52 (Cell Signaling Tech, 4882S), and p65 (Cell Signaling Tech, 3035S).

Techniques: RNA Sequencing, Variant Assay, Gene Expression, Expressing

Journal: Cell Death Discovery

Article Title: Epigenetic context defines the transcriptional activity of canonical and noncanonical NF-κB signaling in pancreatic cancer

doi: 10.1038/s41420-026-03019-9

Figure Lengend Snippet: A Western blot analysis of p105/p50, p100/p52, phospho-RELA, and RELB following TNFα and TWEAK treatments across multiple time points (0–30 min and 1–72 h). Representative of n = 3 independent experiments. B Heatmap of differentially expressed genes following RNA-seq analysis of L3.6pl cells treated with TNFα and TWEAK for 6 and 48 hours. Unbiased clustering analysis was performed after differential expression with DESeq2. One TNFα-treated replicate (replicate 1) was excluded due to low read depth and outlier behavior in PCA and z-score heatmap analyses, which indicated a sequencing error. C Venn diagram displaying the number of TNFα-specific, TWEAK-specific, and commonly differentially expressed genes (DEGs), derived from DESeq2 analysis and unbiased clustering analysis. Selections were based on Log2FC ≥ 1, FDR ≤ 0.05, and base mean ≥ 10. D Pathway analysis for biological processes (GO) and KEGG of TNFα-specific (top) and common genes (bottom) identified in ( C ). The top pathways were selected based on FDR values. E Caspase 3/7 activity in L3.6pl and AsPC-1 cells over 72 h following treatment with TNFα (10 ng/ml), TWEAK (10 ng/ml), and cycloheximide (CHX; 10 µM) as indicated. Values represent total green integrated intensity (normalized to vehicle control and cell number at each time point). Area under the curve (AUC) was calculated for each condition, and p -values were determined using unpaired t -tests based on AUC values. n.s. = not significant. F Single-cell migration tracking assay captured every 15 min over 48 h in AsPC-1 and L3.6pl cells using IncuCyte live-cell imaging (10× magnification). Boxplots depict the mean instantaneous speed calculated from migrated tracks for AsPC-1 and L3.6pl cells following vehicle, TNFα, and TWEAK treatments ( p -value: one-way ANOVA; Dunnett’s multiple comparisons relative to vehicle control). G Scatter plot of normalized expression levels of TNFα and TWEAK (log scale) in 173 PDAC patient samples from TCGA. Each dot represents an individual sample. Based on TNFα and TWEAK expression thresholds (TNFα ≥ 100 and TWEAK < 1300), samples were classified as TNFα high (red), TWEAK high (blue; TNFα < 100 and TWEAK ≥ 1300), TNFα/TWEAK low (purple; TNFα < 60 and TWEAK < 700 or not assigned (black). H Gene Set Enrichment Analysis (GSEA) results for TNFα-specific ( n = 310) and common (n = 626; 575 common genes + 51 TWEAK-specific genes) gene sets in the TNFα high , TWEAK high , and TNFα/TWEAK low expression from TCGA data. Normalized Enrichment Scores (NES) and False Discovery Rate (FDR) values are shown in each plot. TNFα high samples show significant enrichment of both TNFα-specific and common gene sets, whereas TWEAK high samples only display enrichment of the TNFα-specific gene set. I Boxplots showing expression of selected genes from TNFα-specific and common gene sets in PDAC samples grouped by TNFα high , TWEAK high , and TNFα/TWEAK low status. Genes were selected based on their rankings from the GSEA analysis. TNFα-specific genes (top panels) show higher expression in TNFα high samples, while common genes (bottom panels) are elevated in both TNFα high and TWEAK high samples. p -values are shown for significant differences (unpaired t-tests).

Article Snippet: The following primary antibodies were used at the indicated dilutions: phospho-NFκB p65 (S536) (93H1) (phospho-RELA; 1:1000; Cell Signaling Technology; 3033; RRID:AB_331284), RELB (D7D7W) (1:1000; Cell Signaling Technology; 10544; RRID:AB_2797727), NF-κB2 p100/p52 (18D10) (1:1000; Cell Signaling Technology; 3017; RRID:AB_10697356), NF-κB1 p105/p50 (D4P4D) (1:1000; Cell Signaling Technology; 13586; RRID:AB_2665516), and β-Actin (D6A8) (1:1000; Cell Signaling Technology; 8457; RRID:AB_10950489).

Techniques: Western Blot, RNA Sequencing, Quantitative Proteomics, Sequencing, Derivative Assay, Activity Assay, Control, Single Cell, Migration, Live Cell Imaging, Expressing

Journal: Cell Death & Disease

Article Title: CCL11 promotes hepatocellular carcinoma recurrence after surgery by potentiating immunosuppressive CCR5 + CD206 + M2-like macrophages and promoting tumor invasiveness

doi: 10.1038/s41419-026-08508-4

Figure Lengend Snippet: A Flow cytometry quantification of the number of monocyte-differentiated CCR5 + CD206 + M2-like macrophages after administration of recombinant human CCL11 (rhCCL11) protein. Control group: PBS treatment. B The expression Levels of M2-macrophage-related immunosuppressive markers and cytokines in CCL11-treated and PBS-treated CCR5 + M2-like macrophages. C Cytokine array analysis of secreted cytokines in CCL11-treated and PBS-treated CCR5 + M2-like macrophages. Red dot boxes indicate two highly upregulated cytokines: 1, MCP1; 2, IL-8. D mRNA Levels of IL-8 and MCP-1 in CCL11-treated and PBS-treated CCR5 + M2-like macrophages. E CCL11-treated CCR5 + M2-like macrophages enhanced the induction of CD4+ Foxp3+Tregs from naive CD4 + T compared to the PBS-treated control group. F Top 10 enriched KEGG pathways from differentially expressed genes in the CCL11-treated CCR5 + M2-like macrophages. G Gene set enrichment analysis of Toll-like receptor signaling pathway. H Levels of p-IKKα/β, p-IkBα, NFkB1-P50, and PD-L1 expression via western blot analysis. I Schematic diagram of the molecular mechanism of CCL11 in regulating CCR5 + M2-like macrophages. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns not significant.

Article Snippet: Antibodies used for Western blot analysis anti-human/mouse p-IKKα/β (#2078T), anti-human/mouse p-IkBα (#2859T), anti-human/mouse NFKB1-p50 (#13586S), anti-human/mouse p-AKT (#9271S), and anti-human/mouse AKT (#9272S) from Cell Signaling Technology; anti-human/mouse PDL1 (#14-5983-82), anti-human/mouse p-PI3K (#PA5-104853), and anti-human/mouse Mafk (#PA5-115534) from Thermo Fisher Scientific; anti-human/mouse PI3K (#sc-423) and anti-human GAPDH (#sc47724) from Santa Cruz Biotechnology; and anti-human/mouse MMP13 (#18165-1-AP) and anti-mouse GAPDH (#60004-1-Ig) from Proteintech Group.

Techniques: Flow Cytometry, Recombinant, Control, Expressing, Western Blot

Journal: Science Advances

Article Title: Accessory microRNA byproducts expand RNA interference via microprocessor-mediated cleavage activation

doi: 10.1126/sciadv.adz8331

Figure Lengend Snippet: ( A ) Schematics and corresponding predicted RNA folding of engineered microRNA sequences. Each component is labeled and color-coded accordingly. The black arrow represents the site of Drosha cleaved along the miR-7 stem sequence. ( B ) microRNA expression in HEK293 cells infected with different constructs. Bars represent mean values + SD; n = 3 biological replicates. Statistical analysis was performed by two-way ANOVA compared with SCR with Dunnett’s multiple comparisons test. ( C ) Polyacrylamide gel (4 to 20%) Northern blot from whole cell RNA of HEK293 cells transfected with different constructs. ( D ) Rationale for the design of a new microRNA-based platform. Mi-dAp50, microRNA-driven aptamer anti-p50. ( E ) PCR analysis of Mi-dAp50 in samples after RIP by p50 versus immunoglobulin G bait. Bars represent mean values ± SD; n = 3 biological replicates. Statistical analyses were performed by Student’s t test, two tails. ( F ) RT-qPCR showing gene expression level of known NF-κB target genes. RNA was isolated from HEK293 cells expressing different constructs. Bars represent mean values + SD; n = 3 biological replicates. Each comparison was analyzed with Student’s t test, two tails. ( G ) Luminescence detection in HEK293 expressing NF-κB responsive luciferase reporter genes and transfected with different clusters and specific, in presence or absence of TNF-α (10 ng/ml). Parallel experiments were done in Drosha WT and Drosha KO models. Bars represent mean values ± SD; n = 3 biological replicates. Statistical analysis was performed by Student’s t test, two tails. RLU, relative luminescence unit. ( H ) ChIP-PCR of p50 precipitated DNA from HEK293 cells expressing different constructs. The drawing clarifies IKBα as a known NF-κB responsive gene, while RLP30 is used as a negative control. Bars represent mean values ± SD; n = 3 biological replicates. Statistical analysis was performed by one-way ANOVA. * P < 0.05; ** P = < 0.01; *** P = < 0.001; **** P < 0.0001; ns, nonsignificant. TSS, transcription start site.

Article Snippet: Equal volumes of chromatin were immunoprecipitated with either antibody against p50 (catalog no. 13586, Cell Signaling) or rabbit immunoglobulin G (catalog no. P120-101, Bethyl Laboratories, Montgomery, TX, USA) as negative control.

Techniques: Labeling, Sequencing, Expressing, Infection, Construct, Northern Blot, Transfection, Quantitative RT-PCR, Gene Expression, Isolation, Comparison, Luciferase, Negative Control