Journal: Cell Death & Disease

Article Title: C9-ALS/FTD-linked proline–arginine dipeptide repeat protein associates with paraspeckle components and increases paraspeckle formation

doi: 10.1038/s41419-019-1983-5

Figure Lengend Snippet: a The number of EGFP- or EGFP-FLAG-PR100 (EGFP-F-PR100)-expressing paraspeckle-positive NSC34 cells in RNA FISH assay was counted (100 cells/count). The data are presented as means ± SD ( N = 3). Statistical analysis was performed by the unpaired t -test. b, c NSC-34 cells were infected with indicated adenovirus vectors at a multiplicity of infection (MOI) of 800. At 48 h after the infection, the quantitative real-time PCR analysis of NEAT1 was performed ( b ). The cell lysates were subjected to dot blotting analysis using indicated antibodies ( c ). The data are presented as means ± SD ( N = 3). Statistical analysis was performed by one-way analysis of variance (ANOVA) followed by the Dunnett’s multi comparisons test. d , e Primary cultured cerebral cortical neurons (PCNs) were infected with adenovirus encoding FLAG-PR100 at MOIs of 0–200. To keep the constant total MOIs of adenoviruses, appropriate MOIs of LacZ-encoding adenovirus were added for each infection. At 120 h after the infection, the quantitative real-time PCR analysis of NEAT1 was performed ( d ). The cell lysates were subjected to dot blotting analysis using indicated antibodies ( e ). The data are presented as means ± SD ( N = 3). Statistical analysis was performed by one-way ANOVA followed by the Dunnett’s multi comparisons test. f NSC-34 cells were transfected with the NEAT1 -promoter (+) or NEAT1 -promoterless (−) luciferase vector together with the pEF1-Myc/His-vec (−) or the pEF1-FLAG-PR100 (+). At 48 h after the transfection, the luciferase activity was measured. The data are presented as means ± SD ( N = 3). Statistical analysis was performed by one-way ANOVA followed by the Tukey’s multi comparisons test. g , h NSC-34 cells were infected with adenovirus encoding LacZ or mouse NEAT1_1 at an MOI of 1. Cells were also co-infected with adenovirus encoding LacZ or FLAG-PR100 at an MOI of 200 together with adenovirus encoding LacZ (−) or Cre-recombinase (+) at an MOI of 40. At 48 h after the infection, the quantitative real-time PCR analysis of NEAT1 was performed ( g ). The cell lysates were subjected to dot blotting analysis using indicated antibodies ( h ). The data are presented as means ± SD ( N = 3). Statistical analysis was performed by one-way ANOVA followed by the Dunnett’s multi comparisons test. i , j NSC-34 cells were transfected with 0.2 μg/well of the pCMV-GFP or -mouse NEAT1_2 on 6-well plate. After the transfection, NSC-34 cells were infected with adenovirus encoding FLAG-PR100 at an MOI of 200. Total adenoviruses infected were adjusted at an MOI of 400 with adenovirus encoding LacZ. At 48 h after the infection, the quantitative real-time PCR analysis of NEAT1 was performed ( i ). The cell lysates were subjected to dot blotting analysis using indicated antibodies ( j ). The data are presented as means ± SD ( N = 3). Statistical analysis was performed by one-way ANOVA followed by the Dunnett’s multi comparisons test. k , l NSC-34 cells were infected with adenovirus encoding LacZ or FLAG-PR100 at an MOI of 800. At 48 h after the infection, the quantitative real-time PCR analysis of NEAT1, Nfkb1, and Nr4a1 was performed ( k ). The cell lysates were subjected to dot blotting analysis using indicated antibodies ( l ). The data are presented as means ± SD ( N = 3). Statistical analysis was performed by the unpaired t -test. m–o HeLa cells were infected with adenovirus encoding LacZ or FLAG-PR100 at an MOI of 800. At 48 h after the infection, the cell viability was detected by WST-8 assay ( m ). The cell lysates were subjected to dot blotting analysis using indicated antibodies ( n ). The quantitative real-time PCR analysis of NEAT1, CCL5, CXCL8, SH3PXD2A, and TSHZ2 was performed ( o ). The data are presented as means ± SD ( N = 3). Statistical analysis was performed by the unpaired t -test

Article Snippet: Assay IDs of Taqman probes for mouse Neat1, Neat1_2, Hnrnpm, Nfkb1, Nr4a1, and Gapdh are Mm03942186_s1, AJT96WW (Custom Plus TaqMan® RNA Assays), Mm00513070_m1, Mm00476361_m1, Mm01300401_m1, and Mm99999915_g1, respectively.

Techniques: Expressing, Infection, Real-time Polymerase Chain Reaction, Cell Culture, Transfection, Luciferase, Plasmid Preparation, Activity Assay

Journal: Microbiology Spectrum

Article Title: Leishmania donovani Attenuates Dendritic Cell Trafficking to Lymph Nodes by Inhibiting C-Type Lectin Receptor 2 Expression via Transforming Growth Factor-β

doi: 10.1128/spectrum.04122-22

Figure Lengend Snippet: L. donovani -induced TGF-β production suppresses NF-κB-driven CLEC2 upregulation on DCs. (A) Schematic of mouse CLEC1B promoter showing the positions of putative NF-κB-binding site and the primers (P1 and P2) used for ChIP analysis, and details of oligonucleotide probes used for EMSA. Mouse CLEC1B promoter-specific CLEC1B-Pr probe contains putative wild-type NF-κB-binding sites, and MutCLEC1B-Pr probe contains mutations (italicized) at NF-κB-binding site. Base positions are relative to the ATG (translation) start site. (B) EMSA of nuclear extracts of BMDCs treated with LPS for specified times and analyzed with indicated probes. Numbers below lanes indicate densitometry (normalized to OCT-1 binding [internal control]) relative to that of untreated DCs (0 h). Data are representative of three independent experiments (left panel). (Right panel) Relative densitometry results from three separate experiments. (C) Nuclear extracts from BMDCs treated with LPS for 12 h were subjected to EMSA using CLEC1B-Pr and MutCLEC1B-Pr probes, which contained wild-type and mutated NF-κB sites, respectively. Data are representative of those from three independent experiments. (D) Pulldown assay was performed for nuclear proteins derived from LPS-treated (for 12 h) BMDCs using streptavidin (SA)-conjugated Dynabeads and indicated biotin-labeled oligonucleotides. The bound proteins were immunoblotted with antibodies against p65 and p50 subunits of NF-κB. Input represents nuclear extracts (5%) before pulldown. Data are representative of those from three independent experiments. (E) Recruitment of p65 and p50 subunits of NF-κB to the mouse CLEC1B promoter was analyzed (via ChIP-quantitative real-time PCR analysis; primer details are given in Table S1, and the location of primers is indicated in panel A) in BMDCs after treatment with LPS for 12 h. Results are a compilation of those from three experiments ( n = 2 per experiment), presented as fold enrichment relative to that of untreated (UT) BMDCs. (F) BMDCs were transfected with control vector or IκBα dominant negative (IκBαDN) vector or left untransfected (-vec). BMDCs were then treated with LPS for 24 h. The expression of CLEC-2 on BMDCs was analyzed via flow cytometry. Data are representative of three separate analyses (left panel). Right panel: bar graph shows the relative mean fluorescence intensity of CLEC-2 expression (measured as Fig. 1A ) from three separate analyses. (G) BMDCs were transfected or not (-vec) with control or NF-κB p65-encoding vector. Flow cytometry analysis was performed to assess the surface expression of CLEC-2 on BMDCs. Data are representative of three individual experiments (left panel). (Right panel) Combined data of relative mean fluorescence intensity of CLEC-2 expression (calculated as for Fig. 1A ) from three experiments. (H) BMDCs were infected with LDPm for 24 or 48 h or kept uninfected (UI) and then treated (+) or not (−) with LPS for 12 h. EMSA was performed with the indicated probes. Numbers below lanes indicate densitometry of NF-κB binding (normalized to OCT-1 binding). Data are representative of those from three independent experiments (left panel). (Right panel) Relative densitometry data (pooled from three different experiments). (I) Binding of NF-κB to CLEC1B promoter in BMDCs left uninfected or infected with LDPm for 24 h in the absence (−) or presence (+) of isotype control Ab or anti-TGF-β Ab, and then treated (or not) with LPS for 12 h, analyzed via EMSA using indicated probes. Numbers below lanes indicate densitometry (as in panel H), presented relative to uninfected BMDCs that were cultured in the absence of Ab and given no LPS treatment. Data are representative of those from three independent experiments (left panel). (Right panel) Combined densitometry data (relative) from three different experiments. Error bars indicate SD. Each symbol in the graphs corresponds to data derived from one independent experiment (right panels of B, F, G, H, and I) or an individual replicate (E). ***, P < 0.001; **, P < 0.01; *, P < 0.05.

Article Snippet: The same anti-NF-κB p65 and anti-NF-κB p50 antibodies and mouse IgG (sc-2025; from Santa Cruz Biotechnology) were used for ChIP assay.

Techniques: Binding Assay, Control, Derivative Assay, Labeling, Real-time Polymerase Chain Reaction, Transfection, Plasmid Preparation, Dominant Negative Mutation, Expressing, Flow Cytometry, Fluorescence, Infection, Cell Culture

Journal: Microbiology Spectrum

Article Title: Leishmania donovani Attenuates Dendritic Cell Trafficking to Lymph Nodes by Inhibiting C-Type Lectin Receptor 2 Expression via Transforming Growth Factor-β

doi: 10.1128/spectrum.04122-22

Figure Lengend Snippet: TGF-β induced by L. donovani suppresses CLEC-2 expression on DCs via c-Src. (A and B) Immunoblot analysis of the expression of total and phosphorylated (p-) c-Src in lysates of BMDCs infected with LDPm (A) or LDAm (B) for the indicated times. Numbers below lanes indicate densitometry normalized to total c-Src and presented relative to uninfected BMDCs (0 h). Data are representative of those from three experiments (left panel). (Right panel) Relative densitometry data compiled from three experiments. (C) BMDCs were infected with LDPm for 24 h in the presence of the indicated antibodies. The expression of total or phosphorylated c-Src was assessed via immunoblot analysis. Numbers below lanes indicate densitometry (as in panel A) presented relative to uninfected BMDCs cultured in the absence of Ab (no Ab). Data are representative of those from three individual experiments (left panel). (Right panel) Combined densitometry data (relative) from three different experiments. (D) Representative immunoblot (out of three separate experiments) showing the expression of c-Src and β-actin (loading control) in BMDCs left untransfected (-siRNA) or transfected with control siRNA or c-Src-specific siRNA. (E) BMDCs were transfected with siRNAs as for panel D and then infected with LDPm for 24 h or left uninfected and stimulated with LPS for 12 h. The binding of NF-κB to the CLEC1B promoter was determined via EMSA. Numbers below lanes indicate relative densitometry as in Fig. 4H . Data are representative of those from three independent experiments (left panel). (Right panel) Densitometry data pooled from three separate experiments. (F) Flow cytometry analysis of CLEC-2 expression on BMDCs transfected with the indicated siRNAs, then infected with LDPm for 24 h, and stimulated with LPS for another 24 h. Data are representative of three independent experiments (left panel). (Right panel) Relative mean fluorescence intensity of CLEC-2 expression (measured as for Fig. 1A ) from three separate analyses. Error bars indicate SD. Each symbol in the graphs corresponds to data derived from an independent experiment. ***, P < 0.001; **, P < 0.01; *, P < 0.05.

Article Snippet: The same anti-NF-κB p65 and anti-NF-κB p50 antibodies and mouse IgG (sc-2025; from Santa Cruz Biotechnology) were used for ChIP assay.

Techniques: Expressing, Western Blot, Infection, Cell Culture, Control, Transfection, Binding Assay, Flow Cytometry, Fluorescence, Derivative Assay

Journal: Microbiology Spectrum

Article Title: Leishmania donovani Attenuates Dendritic Cell Trafficking to Lymph Nodes by Inhibiting C-Type Lectin Receptor 2 Expression via Transforming Growth Factor-β

doi: 10.1128/spectrum.04122-22

Figure Lengend Snippet: Model illustrating how TGF-β attenuates the lymph node homing capacity of DCs during L. donovani infection. Our results demonstrate a pivotal role of TGF-β and CLEC-2 (a lymph node homing receptor expressed by DCs) in L. donovani -induced impairment of DC migration to lymph nodes (LNs). During L. donovani infection, TGF-β is secreted by DCs. TGF-β then activates c-Src in DCs, which in turn prevents the binding of NF-κB to the CLEC1B (which encodes CLEC-2) promoter and thereby downregulates CLEC-2 expression on DCs (shown within circular inset). The reduced CLEC-2 expression significantly compromises the ability of DCs to migrate from peripheral tissue to draining lymph nodes and hence may attenuate antileishmanial T cell responses. Thus, by limiting the availability of DCs in lymph nodes, CLEC-2 downregulation mediated by the TGF-β/c-Src pathway contributes to immunosuppression during L. donovani infection. The brown-bordered box with the gray shaded area shows the events blocked by L. donovani -induced TGF-β secretion by DCs.

Article Snippet: The same anti-NF-κB p65 and anti-NF-κB p50 antibodies and mouse IgG (sc-2025; from Santa Cruz Biotechnology) were used for ChIP assay.

Techniques: Infection, Migration, Binding Assay, Expressing

Journal: Carcinogenesis

Article Title: Activation of AID by human T-cell leukemia virus Tax oncoprotein and the possible role of its constitutive expression in ATL genesis.

doi: 10.1093/carcin/bgq222

Figure Lengend Snippet: Fig. 5. p50 contributes to AID expression. (A and B) HTLV-1 infection is associated with binding of NF-jB factors to the NF-jB site in AID promoter. Nuclear extracts (5 lg) from control uninfected and HTLV-1-infected T-cell lines (A) and PBMCs from healthy volunteers and patients with ATL (B) were incubated with the labeled double-stranded oligonucleotides representing the AID NF-jB site in EMSA reactions. Nuclear extracts from MT-2 (A, right) and ATL (B, right) cells were subjected to competition analysis with a 100-fold molar excess of unlabeled double-stranded oligonucleotides representing the AID NF-jB site (lane 2), a consensus NF-jB site from the IL-2 receptor a chain (IL-2Ra) promoter (lane 3) or an AP-1 site from the IL-8 promoter (lane 4). The indicated unlabeled oligonucleotides were incubated with nuclear extracts for 15 min before binding reactions. Nuclear extracts from MT-2 cells were also subjected to supershift assays with either no antibody (lane 1) or the indicated antibodies (Abs; lanes 5–9). The Abs were incubated with nuclear extracts for 45 min before binding reactions. Arrows: the specific complexes, arrowheads: the DNA binding complexes supershifted by Abs. (C) Tax-induced NF-jB-binding activity. Nuclear extracts from JPX-9 cells treated with CdCl2 (20 lM) for the indicated time periods were incubated with the labeled double-stranded oligonucleotides representing the AID NF-jB site (top). Nuclear extracts from JPX-9 cells treated with CdCl2 (20 lM) for 4 h were subjected to competition analysis with a 100-fold molar excess of unlabeled double-stranded oligonucleotides representing the AID NF-jB site (lane 3), a consensus NF-jB site from the IL-2a promoter (lane 4) and an AP-1 site from the IL-8 promoter (lane 5). (D) Recombinant NF-jB subunit p50 was subjected to EMSA using the labeled double-stranded oligonucleotides representing the AID NF-jB site. Binding reaction was carried out in the presence of the indicated competitors (lanes 2–5). Lane 1, DNA complex in the absence of any competitor. In addition, incubation with antibody to p50 (lane 6) but not p65 (lane 7) supershifted the complex. Arrows: the specific complexes, arrowheads: the DNA-binding complexes supershifted by Abs. (E) Effect of overexpression of p50 and p65 on AID promoter. Jurkat cells were transfected with an expression plasmid for either p50 or p65 component of NF-jB (5 lg) along with either pGL3 basic or AID 0.9P (5 lg). Luciferase activity was analyzed, and the fold induction of p50 or p65 activity was calculated relative to the empty vector. Data are mean ± SD of three independent transfection experiments. (F) Suppression of endogenous p50 reduces the expression of AID mRNA and NF-jB DNA binding. MT-2 cells were transfected with either p50 or control siRNA and either p65 or control siRNA. At 48 h after transfection, total RNAwas isolated from each cell, and the expression levels of p50, p65, AID and b-actin (loading control) mRNAs were measured by RT–PCR. Nuclear extracts were also isolated from each cell and subjected to EMSA with the AID NF-jB probe (right). (G) Effects of NF-jB inhibitors on endogenous AID expression in an HTLV-1-infected T cell line. MT-2 cells were treated with either Bay 11-7082 (10 lM) or N-acetyl-L-leucyl-L- leucyl-L-norleucinal (20 lM) for the indicated time periods. Total RNA was isolated from each cell, and the AID mRNA expression level was measured by RT–PCR.

Article Snippet: The cells were washed with PBS containing 7% of FBS once and resuspended in PBS/7% FBS containing rabbit polyclonal antibody to Bcl-3 (Santa Cruz Biotechnology) and mouse monoclonal antibody to p50 (Santa Cruz Biotechnology) for 20 min at room temperature.

Techniques: Expressing, Infection, Binding Assay, Control, Incubation, Labeling, Activity Assay, Recombinant, Over Expression, Transfection, Plasmid Preparation, Luciferase, Isolation, Reverse Transcription Polymerase Chain Reaction

Journal: Carcinogenesis

Article Title: Activation of AID by human T-cell leukemia virus Tax oncoprotein and the possible role of its constitutive expression in ATL genesis.

doi: 10.1093/carcin/bgq222

Figure Lengend Snippet: Fig. 6. HTLV-1-infected T cells express Bcl-3/p50 complexes. (A) Expression of Bcl-3 in HTLV-1-infected T-cell lines. RT–PCR analysis was carried out for Bcl- 3 and b-actin (loading control). Western blot analysis was performed for Bcl-3 and actin. (B) Induction of Bcl-3 expression by Tax. JPX-9 cells were treated with or without 20 lM of CdCl2 for the indicated time periods. RT–PCR was carried out for Bcl-3 and b-actin (loading control). Western blot analysis was also performed for Tax, Bcl-3 and actin (loading control). (C) Immunofluorescence images of MT-2 cells for p50 (Alexa Fluor 488; green), Bcl-3 (Alexa Fluor 546; red) and nuclei (Hoechst 33342; blue). p50 colocalizes with Bcl-3 in situ in the nuclei of MT-2 cells; DIC, differential interference contrast. (D) Cell lysates from MT-2 cells were used for immunoprecipitation with anti-Bcl-3 or anti-p50 antibody followed by immunoblotting with anti-p50 or anti-Bcl-3 antibody. The expression level of p50 or Bcl-3 was detected (input). (E) RT–PCR analysis for AID, Bcl-3 and Tax expression in primary ATL cells.

Article Snippet: The cells were washed with PBS containing 7% of FBS once and resuspended in PBS/7% FBS containing rabbit polyclonal antibody to Bcl-3 (Santa Cruz Biotechnology) and mouse monoclonal antibody to p50 (Santa Cruz Biotechnology) for 20 min at room temperature.

Techniques: Infection, Expressing, Reverse Transcription Polymerase Chain Reaction, Control, Western Blot, In Situ, Immunoprecipitation

Journal: Cell Reports Medicine

Article Title: Oncostatin M signaling drives cancer-associated skeletal muscle wasting

doi: 10.1016/j.xcrm.2024.101498

Figure Lengend Snippet:

Article Snippet: NF-κB1 p105/p50 (D7H5M) Rabbit Antibody , Cell Signaling , Cat# 12540; RRID: AB_2687614.

Techniques: Ubiquitin Proteomics, Virus, Recombinant, SYBR Green Assay, Transfection, Expressing, Purification, Enzyme-linked Immunosorbent Assay, Plasmid Preparation, Software

Journal: Carcinogenesis

Article Title: Activation of AID by human T-cell leukemia virus Tax oncoprotein and the possible role of its constitutive expression in ATL genesis.

doi: 10.1093/carcin/bgq222

Figure Lengend Snippet: Fig. 5. p50 contributes to AID expression. (A and B) HTLV-1 infection is associated with binding of NF-jB factors to the NF-jB site in AID promoter. Nuclear extracts (5 lg) from control uninfected and HTLV-1-infected T-cell lines (A) and PBMCs from healthy volunteers and patients with ATL (B) were incubated with the labeled double-stranded oligonucleotides representing the AID NF-jB site in EMSA reactions. Nuclear extracts from MT-2 (A, right) and ATL (B, right) cells were subjected to competition analysis with a 100-fold molar excess of unlabeled double-stranded oligonucleotides representing the AID NF-jB site (lane 2), a consensus NF-jB site from the IL-2 receptor a chain (IL-2Ra) promoter (lane 3) or an AP-1 site from the IL-8 promoter (lane 4). The indicated unlabeled oligonucleotides were incubated with nuclear extracts for 15 min before binding reactions. Nuclear extracts from MT-2 cells were also subjected to supershift assays with either no antibody (lane 1) or the indicated antibodies (Abs; lanes 5–9). The Abs were incubated with nuclear extracts for 45 min before binding reactions. Arrows: the specific complexes, arrowheads: the DNA binding complexes supershifted by Abs. (C) Tax-induced NF-jB-binding activity. Nuclear extracts from JPX-9 cells treated with CdCl2 (20 lM) for the indicated time periods were incubated with the labeled double-stranded oligonucleotides representing the AID NF-jB site (top). Nuclear extracts from JPX-9 cells treated with CdCl2 (20 lM) for 4 h were subjected to competition analysis with a 100-fold molar excess of unlabeled double-stranded oligonucleotides representing the AID NF-jB site (lane 3), a consensus NF-jB site from the IL-2a promoter (lane 4) and an AP-1 site from the IL-8 promoter (lane 5). (D) Recombinant NF-jB subunit p50 was subjected to EMSA using the labeled double-stranded oligonucleotides representing the AID NF-jB site. Binding reaction was carried out in the presence of the indicated competitors (lanes 2–5). Lane 1, DNA complex in the absence of any competitor. In addition, incubation with antibody to p50 (lane 6) but not p65 (lane 7) supershifted the complex. Arrows: the specific complexes, arrowheads: the DNA-binding complexes supershifted by Abs. (E) Effect of overexpression of p50 and p65 on AID promoter. Jurkat cells were transfected with an expression plasmid for either p50 or p65 component of NF-jB (5 lg) along with either pGL3 basic or AID 0.9P (5 lg). Luciferase activity was analyzed, and the fold induction of p50 or p65 activity was calculated relative to the empty vector. Data are mean ± SD of three independent transfection experiments. (F) Suppression of endogenous p50 reduces the expression of AID mRNA and NF-jB DNA binding. MT-2 cells were transfected with either p50 or control siRNA and either p65 or control siRNA. At 48 h after transfection, total RNAwas isolated from each cell, and the expression levels of p50, p65, AID and b-actin (loading control) mRNAs were measured by RT–PCR. Nuclear extracts were also isolated from each cell and subjected to EMSA with the AID NF-jB probe (right). (G) Effects of NF-jB inhibitors on endogenous AID expression in an HTLV-1-infected T cell line. MT-2 cells were treated with either Bay 11-7082 (10 lM) or N-acetyl-L-leucyl-L- leucyl-L-norleucinal (20 lM) for the indicated time periods. Total RNA was isolated from each cell, and the AID mRNA expression level was measured by RT–PCR.

Article Snippet: Mouse monoclonal antibody to p50 and rabbit polyclonal antibody to Bcl-3 (Santa Cruz Biotechnology) and rabbit polyclonal antibody to p50 (Cell Signaling Technology) were used for immunoprecipitation and western blot.

Techniques: Expressing, Infection, Binding Assay, Control, Incubation, Labeling, Activity Assay, Recombinant, Over Expression, Transfection, Plasmid Preparation, Luciferase, Isolation, Reverse Transcription Polymerase Chain Reaction

Journal: Carcinogenesis

Article Title: Activation of AID by human T-cell leukemia virus Tax oncoprotein and the possible role of its constitutive expression in ATL genesis.

doi: 10.1093/carcin/bgq222

Figure Lengend Snippet: Fig. 6. HTLV-1-infected T cells express Bcl-3/p50 complexes. (A) Expression of Bcl-3 in HTLV-1-infected T-cell lines. RT–PCR analysis was carried out for Bcl- 3 and b-actin (loading control). Western blot analysis was performed for Bcl-3 and actin. (B) Induction of Bcl-3 expression by Tax. JPX-9 cells were treated with or without 20 lM of CdCl2 for the indicated time periods. RT–PCR was carried out for Bcl-3 and b-actin (loading control). Western blot analysis was also performed for Tax, Bcl-3 and actin (loading control). (C) Immunofluorescence images of MT-2 cells for p50 (Alexa Fluor 488; green), Bcl-3 (Alexa Fluor 546; red) and nuclei (Hoechst 33342; blue). p50 colocalizes with Bcl-3 in situ in the nuclei of MT-2 cells; DIC, differential interference contrast. (D) Cell lysates from MT-2 cells were used for immunoprecipitation with anti-Bcl-3 or anti-p50 antibody followed by immunoblotting with anti-p50 or anti-Bcl-3 antibody. The expression level of p50 or Bcl-3 was detected (input). (E) RT–PCR analysis for AID, Bcl-3 and Tax expression in primary ATL cells.

Article Snippet: Mouse monoclonal antibody to p50 and rabbit polyclonal antibody to Bcl-3 (Santa Cruz Biotechnology) and rabbit polyclonal antibody to p50 (Cell Signaling Technology) were used for immunoprecipitation and western blot.

Techniques: Infection, Expressing, Reverse Transcription Polymerase Chain Reaction, Control, Western Blot, In Situ, Immunoprecipitation

Journal: Journal of dairy science

Article Title: Syndecan-3 inhibits LPS-induced Inflammation of Bovine Mammary Epithelial Cells through the NF-κB Signal Transduction Pathway.

doi: 10.3168/jds.2024-25212

Figure Lengend Snippet: Figure 4. SDC3 inhibits LPS-induced inflammatory response in BMECs through the NF-κB signaling pathway. The cells in different experi- mental groups were treated with LPS, the overexpression vector (pBI-CMV3-SDC3), and the overexpression vector + LPS. After total protein was extracted, the protein expression levels of nuclear factor kappa B subunit 1 (NF-κB p50), Phospho-IκB Alpha (p-IκBα), and IκBα in BMECs from each experimental group were analyzed by Western blot (A). Western blot images were analyzed using ImageJ software, and the expression of the studied proteins was normalized to that of β-actin (B-D). Data are presented as the mean ± SD (n = 3). Significant differences are indicated by * P < 0.05, ** P < 0.01.

Article Snippet: The information of primary antibodies is listed as follows: β-actin (1:5000, AP0060, Bioworld Technology, China); Rabbit anti-SDC3 antibody(1:5000, bs-15503R, Bioss, China); IκBα antibody (1:5000, 10268–1-AP, Proteintech, China), p-IκBα antibody (1:1000, bs-2513R, Bioss, China), NF-κB p50 antibody (1:5000, 14220–1-AP, Proteintech, China).

Techniques: Over Expression, Plasmid Preparation, Expressing, Western Blot, Software

Journal: Journal of dairy science

Article Title: Syndecan-3 inhibits LPS-induced Inflammation of Bovine Mammary Epithelial Cells through the NF-κB Signal Transduction Pathway.

doi: 10.3168/jds.2024-25212

Figure Lengend Snippet: Figure 5. Summary of the mechanism by which SDC3 inhibits LPS- induced cellular inflammation. The overexpression of SDC3 inhibits the expression of NF-κB p50 and p-IκBα, increases the expression of IκBα, inhibits the activation of NF-κB signal pathway induced by LPS, thus reduces the transcription of inflammatory cytokines (IL-6, IL-1β, TNFα) mediated by NF-κB, and finally inhibits the LPS-induced inflammation of BMECs.

Article Snippet: The information of primary antibodies is listed as follows: β-actin (1:5000, AP0060, Bioworld Technology, China); Rabbit anti-SDC3 antibody(1:5000, bs-15503R, Bioss, China); IκBα antibody (1:5000, 10268–1-AP, Proteintech, China), p-IκBα antibody (1:1000, bs-2513R, Bioss, China), NF-κB p50 antibody (1:5000, 14220–1-AP, Proteintech, China).

Techniques: Over Expression, Expressing, Activation Assay

Journal:

Article Title: Role of the NF-?B Signaling Pathway and ?B cis -Regulatory Elements on the IRF-1 and iNOS Promoter Regions in Mycobacterial Lipoarabinomannan Induction of Nitric Oxide

doi: 10.1128/IAI.71.3.1442-1452.2003

Figure Lengend Snippet: ManLAM activates functional NF-κB. (A) RAW 264.7γNO(−) cells were stimulated with ManLAM (10 μg/ml) for 30 min, and nuclear proteins were isolated, followed by an EMSA with a 32P-labeled NF-κB consensus oligonucleotide. Compared to unstimulated cells, ManLAM induced NF-κB activation. The specificity of the NF-κB complex was verified by coincubation of the nuclear extract-oligonucleotide mixture with an anti-p50 antibody (α-p50). The nonspecific (NS) band was present in both unstimulated and ManLAM-stimulated cells and did not supershift with the anti-p50 antibody. (B) Unstimulated and ManLAM-stimulated RAW 264.7γNO(−) cells were stained with DAPI for nuclear detection and immunostained with an anti-p50-NF-κB antibody. The cells were then viewed under a fluorescent microscope at selective wavelengths to detect nuclear staining (i and iv), p50-NF-κB staining (ii and v), or both (iii and vi). After counting of 200 cells in random fields, the percentage of cells with positive nuclear staining for p50-NF-κB was 12.5% for unstimulated cells and 46.3% for ManLAM-stimulated cells. (C) RAW 264.7γNO(−) cells were transfected with 2 μg of the κB-SEAP plasmid. At 0 and 24 h after stimulation with) ManLAM (10 μg/ml), the supernatant was assayed for alkaline phosphatase activity (*, P < 0.05; **, P < 0.01; ***, P < 0.001). Data shown in panels A and B are representative of three independent experiments. Data shown in panel C are means ± SD from three independent experiments.

Article Snippet: Mouse IFN-γ was obtained from R & D Systems Inc, Minneapolis, Minn. A rabbit anti-iNOS polyclonal antibody was purchased from Alexis Biochemicals, San Diego, Calif. Anti-p50 NF-κB, anti-c-Jun, anti-c-Fos, and anti-IRF-1 antibodies were purchased from Santa Cruz Biotechnology, Santa Cruz, Calif.

Techniques: Functional Assay, Isolation, Labeling, Activation Assay, Staining, Microscopy, Transfection, Plasmid Preparation, Activity Assay

Journal:

Article Title: Role of the NF-?B Signaling Pathway and ?B cis -Regulatory Elements on the IRF-1 and iNOS Promoter Regions in Mycobacterial Lipoarabinomannan Induction of Nitric Oxide

doi: 10.1128/IAI.71.3.1442-1452.2003

Figure Lengend Snippet: (A and B) Both the basal κBI and the enhancer κBII site on the 5′-flanking region of the iNOS promoter bind ManLAM-induced NF-κB. Macrophages were stimulated with ManLAM (10 μg/ml) for 30 min, and nuclear protein was isolated and probed with 32P-labeled oligonucleotides containing either the κBI (A) or the κBII (B) site. As shown, both the κBI and κBII sites bind NF-κB, as confirmed by supershift of the NF-κB-containing complexes with the anti-p50 antibody (α-p50). (C) Stimulation with IFN-γ (10 U/ml) did not induce NF-κB binding to either the κBI or the κBII site. (D) Moreover, neither IFN-γ-induced Stat1α nor IFN-γ-induced IRF-1 bound either NF-κB-binding site, although, as expected, there was binding to GAS and ISRE sites, respectively. Data shown are representative of two independent experiments. Slashed circles indicate no nuclear extract in the binding reaction; minus signs indicate unstimulated cells.

Article Snippet: Mouse IFN-γ was obtained from R & D Systems Inc, Minneapolis, Minn. A rabbit anti-iNOS polyclonal antibody was purchased from Alexis Biochemicals, San Diego, Calif. Anti-p50 NF-κB, anti-c-Jun, anti-c-Fos, and anti-IRF-1 antibodies were purchased from Santa Cruz Biotechnology, Santa Cruz, Calif.

Techniques: Isolation, Labeling, Binding Assay

Journal:

Article Title: Role of the NF-?B Signaling Pathway and ?B cis -Regulatory Elements on the IRF-1 and iNOS Promoter Regions in Mycobacterial Lipoarabinomannan Induction of Nitric Oxide

doi: 10.1128/IAI.71.3.1442-1452.2003

Figure Lengend Snippet: Both the basal and enhancer NF-κB-binding sites on the 5′-flanking region of the iNOS promoter are required for IFN-γ plus ManLAM-induction of iNOS. RAW 264.7γNO(−) cells were transfected with 0.3 μg of either the iNOS-luc plasmid, the mut-κBI-iNOS-luc plasmid, the mut-κBII-iNOS-luc plasmid, or the mut-κBI-mut-κBII-iNOS-luc plasmid. After 8 h of stimulation with IFN-γ (10 U/ml) plus ManLAM (10 μg/ml), nucleus-free lysates were assayed for luciferase activity. Results are reported as fold-increase in relative light units (Fold RLU) and normalized for protein concentration. Data are means ± SD from three independent experiments. **, P < 0.01; ***, P < 0.001 (comparisons to second bar from left).

Article Snippet: Mouse IFN-γ was obtained from R & D Systems Inc, Minneapolis, Minn. A rabbit anti-iNOS polyclonal antibody was purchased from Alexis Biochemicals, San Diego, Calif. Anti-p50 NF-κB, anti-c-Jun, anti-c-Fos, and anti-IRF-1 antibodies were purchased from Santa Cruz Biotechnology, Santa Cruz, Calif.

Techniques: Binding Assay, Transfection, Plasmid Preparation, Luciferase, Activity Assay, Protein Concentration

Journal:

Article Title: Role of the NF-?B Signaling Pathway and ?B cis -Regulatory Elements on the IRF-1 and iNOS Promoter Regions in Mycobacterial Lipoarabinomannan Induction of Nitric Oxide

doi: 10.1128/IAI.71.3.1442-1452.2003

Figure Lengend Snippet: ManLAM-induced NF-κB binds to the 5′-flanking region of the IRF-1 promoter but does not induce IRF-1 expression. (A) RAW 264.7γNO(−) cells were stimulated with ManLAM for 30 min, and nuclear extracts were probed with a 32P-labeled oligonucleotide that spans the NF-κB-binding site on the 5′-flanking region of the IRF-1 promoter. As shown, ManLAM-induced NF-κB binds to this site and was supershifted with the anti-p50 antibody (α-p50). (B) To determine if ManLAM is capable of inducing IRF-1 expression, the cells were stimulated with either 10 or 50 μg of ManLAM/ml for 2 or 18 h, followed by Western blotting for IRF-1 (lanes 8 to 11). For positive controls, the cells were stimulated with IFN-γ (10 U/ml) for 2 and 18 h (lanes 2 and 3). Since RAW 264.7 cells are quite sensitive to LPS, they were also stimulated with 1 and 100 ng of LPS/ml for 2 and 18 h (lanes 4 to 7). Whereas IFN-γ induced IRF-1 protein expression, neither ManLAM nor LPS did, even at relatively high concentrations. (C) RAW264.7γNO(−) cells stimulated with IFN-γ or ManLAM at the indicated concentrations and times were lysed with Laemmli sample buffer containing 2.5% SDS, which lyses both plasma and nuclear membranes; the lysates were then sonicated for 2 s with a probe sonicator, separated by SDS-PAGE, and immunoblotted for IRF-1. (D) Nuclear proteins were isolated from unstimulated cells and from cells stimulated with IFN-γ, LPS, or ManLAM at the indicated concentrations and times. The nuclear proteins were then separated by SDS-PAGE and immunoblotted for IRF-1.

Article Snippet: Mouse IFN-γ was obtained from R & D Systems Inc, Minneapolis, Minn. A rabbit anti-iNOS polyclonal antibody was purchased from Alexis Biochemicals, San Diego, Calif. Anti-p50 NF-κB, anti-c-Jun, anti-c-Fos, and anti-IRF-1 antibodies were purchased from Santa Cruz Biotechnology, Santa Cruz, Calif.

Techniques: Expressing, Labeling, Binding Assay, Western Blot, Sonication, SDS Page, Isolation

Journal:

Article Title: Role of the NF-?B Signaling Pathway and ?B cis -Regulatory Elements on the IRF-1 and iNOS Promoter Regions in Mycobacterial Lipoarabinomannan Induction of Nitric Oxide

doi: 10.1128/IAI.71.3.1442-1452.2003

Figure Lengend Snippet: ManLAM-induced NF-κB is unaffected by the MAPKs and does not appear to associate with c-Jun or c-Fos. (A) RAW 264.7γNO(−) cells were either left unstimulated, stimulated with ManLAM (10 μg/ml), or pretreated with PD98059, SB203580, or the JNK inhibitor (JNKi), followed by stimulation with ManLAM (10 μg/ml) for 2 h. Nuclear proteins were isolated, and EMSA was performed on the nuclear extracts by using a 32P-end-labeled NF-κB oligonucleotide. (B) The ManLAM-stimulated NF-κB-oligonucleotide complex is supershifted with the p50 antibody but not with either a c-Jun or a c-Fos antibody.

Article Snippet: Mouse IFN-γ was obtained from R & D Systems Inc, Minneapolis, Minn. A rabbit anti-iNOS polyclonal antibody was purchased from Alexis Biochemicals, San Diego, Calif. Anti-p50 NF-κB, anti-c-Jun, anti-c-Fos, and anti-IRF-1 antibodies were purchased from Santa Cruz Biotechnology, Santa Cruz, Calif.

Techniques: Isolation, Labeling

Journal: Oncogene

Article Title: Inhibition of NF-kappaB sensitizes human pancreatic carcinoma cells to apoptosis induced by etoposide (VP16) or doxorubicin.

doi: 10.1038/sj.onc.1204168

Figure Lengend Snippet: Figure 2 NF-kB binding activity prior to and during treatment with VP16. BxPc-3, PT45-P1, Capan-1 and A818-4 cells were exposed to VP16 (20 mM) for various periods. (a) At the indicated time nuclear protein was prepared for gel-shift assays performed with a 32P-labeled NF-kB consensus oligonucleotides as probe. Nuclear extracts from untreated cells were submitted to: (b) supershift assays with monoclonal antibodies against p65 and p50 or against AP-1 as control; or to (c) gel-shift assays performed with 32P-labeled NF-kB or AP-1 consensus oligonucleotides. Representative results from three independent experiments are shown

Article Snippet: For NF-kB supershift assay, anti p65/ p50 NF-kB antibodies (Santa Cruz; Heidelberg, Germany) were added (1 h, 48C).

Techniques: Binding Assay, Activity Assay, Gel Shift, Labeling, Bioprocessing, Control

Journal: Oncogene

Article Title: Inhibition of NF-kappaB sensitizes human pancreatic carcinoma cells to apoptosis induced by etoposide (VP16) or doxorubicin.

doi: 10.1038/sj.onc.1204168

Figure Lengend Snippet: Figure 3 Inhibition of basal and VP16-induced NF-kB activity by gliotoxin, MG132 and sulfasalazine. (a) Upon transfection with NF-kB- or AP-1 driven luciferase reporter gene vectors, pancreatic carcinoma cells were treated with VP16 (20 mM) for 6 h, or not, in the absence or presence of 10 mM MG132 [M], 0.5 mM gliotoxin [G] and 0.5 mM sulfasalazine [S]. NF-kB- and AP-1-dependent luciferase expression was calculated as arbitrary units. Data express the mean+s.d. of four independent experiments. (b) Pancreatic carcinoma cells were treated with PV16 (20 mM) for 3 h, or not, in the absence or presence of MG132, gliotoxin and sulfasalazine. Nuclear protein was prepared for gel-shift assays performed using a 32P-labeled NF-kB consensus oligonucleotides as probe. A representative result from three independent experiments is shown

Article Snippet: For NF-kB supershift assay, anti p65/ p50 NF-kB antibodies (Santa Cruz; Heidelberg, Germany) were added (1 h, 48C).

Techniques: Inhibition, Activity Assay, Transfection, Luciferase, Expressing, Gel Shift, Labeling

Journal: Oncogene

Article Title: Inhibition of NF-kappaB sensitizes human pancreatic carcinoma cells to apoptosis induced by etoposide (VP16) or doxorubicin.

doi: 10.1038/sj.onc.1204168

Figure Lengend Snippet: Figure 5 Expression of the IkBa super-repressor blocks NF-kB activity. Capan-1 and A818-4 cells were transiently transfected with an expression vector for DN-IkBa or lacZ (mock). (a) DN- IkBa (lower panel) or lacZ (upper panel) transfectants were exposed to VP16 (20 mM) or TNFa (0.2 nM) for 20 and 120 min and were analysed for IkBa/DN-IkBa expression by Western blotting; a representative result from four independent experi- ments is shown. (b) Transfectants were treated with VP16 (20 mM) for 3 h, or without, and were analysed for NF-kB binding activity by gel-shift assay; a representative result from three independent experiments is shown. (c) Transfectants were treated with VP16 (20 mM) for 8 h, or without, and were analysed for luciferase expression driven by NF-kB. For control, AP-1 driven luciferase expression was determined. Results express the mean+s.d., n=4

Article Snippet: For NF-kB supershift assay, anti p65/ p50 NF-kB antibodies (Santa Cruz; Heidelberg, Germany) were added (1 h, 48C).

Techniques: Expressing, Activity Assay, Transfection, Plasmid Preparation, Western Blot, Binding Assay, Gel Shift, Luciferase, Control

Journal: Oncogene

Article Title: Cytoplasmic dynein regulates the subcellular localization of sphingosine kinase 2 to elicit tumor-suppressive functions in glioblastoma

doi: 10.1038/s41388-018-0504-9

Figure Lengend Snippet: SK2 interacts with dynein IC. a HEK293 cells were transfected with empty vector (EV) or a vector encoding HA-tagged IC2, either individually or in combination with a vector encoding FLAG-tagged SK2. SK2 was immunoprecipitated from cell lysates with anti-FLAG antibodies, and co-immunoprecipitated IC2 was detected by immunoblotting with anti-HA antibodies. Expression levels of IC2 in the lysates were confirmed by immunoblotting with anti-HA antibodies (Lysate). Immunoprecipitates were also probed with anti-FLAG antibodies to confirm the presence of SK2. Blots shown are representative of at least five independent experiments. b HEK293 cells were transfected with empty vector (EV) or a vector encoding FLAG-tagged SK2, either individually or in combination with a vector encoding HA-tagged IC1 or HA-tagged IC2. Lysates were pre-cleared with Protein G µbeads. Co-immunoprecipitation and immunoblotting analyses were then performed as described in ( a ). Blots shown are representative of three independent experiments. c SK2 was immunoprecipitated from murine whole brain lysate using anti-SK2 antibodies. Co-immunoprecipitated dynein intermediate chains (IC), light intermediate chain 1 (LIC1) and dynactin p150 were detected by immunoblotting with anti-IC, anti-LIC1 and anti-dynactin p150 antibodies, respectively. Expression levels of these proteins in the mouse brain lysate were confirmed by immunoblot analyses with their respective antibodies (Lysate). Lysates and immunoprecipitates were also probed with anti-SK2 antibodies to confirm expression and immunoprecipitation of SK2. H/C designates the heavy chain IgG band. Blots shown are representative of three independent experiments. d Immunofluorescence staining and confocal microscopy demonstrating co-localization of SK2 and dynein IC in HEK293 cells. SK2 (green) was detected using anti-SK2 antibodies and dynein IC (red) was detected using anti-IC antibodies. Nuclei were highlighted using DAPI (blue). Images are representative of at least 100 cells, from three independent experiments. Scale bar = 10 μm. e Immunofluorescence analysis and confocal microscopy demonstrating direct interactions between SK2 and dynein IC, using the Duolink® in situ PLA system with anti-SK2 (1:300; ECM Biosciences) and anti-IC antibodies (1:300) in HEK293 cells (top panels). Each red dot indicates a single direct interaction. Nuclei were highlighted using DAPI (blue). Differential interference contrast images are also shown (bottom panels). Images are representative of at least 100 cells, from three independent experiments. Scale bar = 10 μm

Article Snippet: The following primary antibodies were utilized: anti-FLAG (Clone M2 #F3165, Sigma-Aldrich), anti-HA (#H3663, Sigma-Aldrich), anti-DYNC1IC (clone 74.1, #MAB1618, Millipore), anti-dynactin p150 (#SC-135890, Santa Cruz Biotechnology), anti-α-tubulin (#ab7291, Abcam), rabbit anti-DYNC1I1 (#13808-1-AP, Proteintech), anti-DYNC1I2 (#ab96288, Abcam), anti-DYNC1LIC1 (#ab157468, Abcam), anti-SK2 (#SP4621, ECM Biosciences; and #17096-1-AP, Proteintech), anti-GFP (#600-101-215, Rockland Immunochemicals), anti-PECAM-1 (CD31; #SC-1506, Santa Cruz Biotechnology) and anti-Ki67 (#VP-K452, Vector Laboratories).

Techniques: Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot, Expressing, Immunofluorescence, Staining, Confocal Microscopy, In Situ

Journal: Frontiers in immunology

Article Title: Detrimental NFKB1 missense variants affecting the Rel-homology domain of p105/p50.

doi: 10.3389/fimmu.2022.965326

Figure Lengend Snippet: FIGURE 1 Missense variants analyzed in this study and domain structure of p105/p50 (Upper panel) Amino acid changes localizing to the N-terminal half of p105 affect both the precursor and the mature p50. Blue, variants tested in p105 and p50; black, variants tested in p50 only. The panel comprises all p50 variants enrolled in the Tuijnenburg and Lorenzini studies, except R231H (underlined). The deleterious variant Y350C has previously been described (23) and was included as a prototypical control. (Lower panel) The protein domain structure of the p105 precursor (long horizontal arrow) with the Rel-homology domain (RHD; red), glycine-rich region (GRR; blue), Ankyrin-repeat domain (ANK; yellow) and death domain (DD; green). Removal of the C-terminal half by limited proteolysis generates the mature transcription factor subunit p50 (short horizontal arrow). Numbers denominate amino acid positions. The position of the nuclear localization sequence (NLS) is indicated by an arrow.

Article Snippet: Primary antibodies were rabbit-anti-NF-kB1 #13586 (raised against residues surrounding Ile415 of mouse NF-kB1 to simultaneously detect p105 and p50) and mouseanti-beta-actin #3700 (both from Cell Signaling; NEB; Frankfurt, Germany).

Techniques: Variant Assay, Control, Sequencing

Journal: Frontiers in immunology

Article Title: Detrimental NFKB1 missense variants affecting the Rel-homology domain of p105/p50.

doi: 10.3389/fimmu.2022.965326

Figure Lengend Snippet: FIGURE 2 Deleterious missense variants causing protein loss are characterized by weak p105 expression and sub-nuclear deposition of p50. HEK293T cells were transiently transfected with expression vectors encoding EGFP-tagged proteins of wildtype or mutant p105 or p50 as indicated and analyzed by fluorescence microscopy. The known devastating Y350C variant was included as a control. Scale bars are indicated. Overlay images with stained nuclei are shown in Supplementary Figures 1A, B. Representative results are shown. (A) Ectopically expressed p105 predominantly localizes to the cytoplasm. EGFP-fused wildtype p105 and non-decaying variants yield robust expression levels. Limited expression and/or aberrant localization indicate severe protein defects (I87S, V98D, R157P, Y286N, W295C, and Y350C). (B) Transiently overexpressed EGFP- tagged wildtype p50 and non-decaying variants show a homogeneous nuclear distribution. Deleterious protein defects are indicated by unusual sub-nuclear protein deposition into aggregate-like structures with high fluorescence intensities (I87S, G92V, V98D, I142T, R157P, R284P, Y286N, W295C and Y350C).

Article Snippet: Primary antibodies were rabbit-anti-NF-kB1 #13586 (raised against residues surrounding Ile415 of mouse NF-kB1 to simultaneously detect p105 and p50) and mouseanti-beta-actin #3700 (both from Cell Signaling; NEB; Frankfurt, Germany).

Techniques: Expressing, Transfection, Mutagenesis, Microscopy, Variant Assay, Control, Staining

Journal: Frontiers in immunology

Article Title: Detrimental NFKB1 missense variants affecting the Rel-homology domain of p105/p50.

doi: 10.3389/fimmu.2022.965326

Figure Lengend Snippet: FIGURE 3 Damaging NFKB1 variants cause rapid p105 decay, abrogate processing of p105 to generate p50, and prohibit a sustained abundance of p50. HEK293T cells were transiently transfected with EGFP-fusion constructs either encoding p105 or p50 missense variants as indicated. Variants depicted in blue font were included in both panels. Whole-cell lysates were analyzed by Western blotting using antibodies directed against an epitope near the C-terminal end of p50, to simultaneously detect p105 and p50 (both green), and against b-actin (red) as loading control. (A) In cells transfected with wildtype p105 or non-decaying p105 mutants, an invariant proportion of the ectopically expressed p105 is converted to p50 by endogenous mechanisms. Deleterious variants are identified by weaker p105 expression and low or undetectable p50 (I87S, V98D, R157P, Y286N, W295C, and Y350C). Representative results of five independent experiments are shown. (B) Upon enforced expression of p50 (skipping the precursor stage) deleterious variants only gain limited expression levels (I87S, G92V, V98D, I142T, R157P, R284P, Y286N, W295C and Y350C). Representative results of six independent experiments are shown. Samples were blotted once in two experiments and at least twice in four experiments.

Article Snippet: Primary antibodies were rabbit-anti-NF-kB1 #13586 (raised against residues surrounding Ile415 of mouse NF-kB1 to simultaneously detect p105 and p50) and mouseanti-beta-actin #3700 (both from Cell Signaling; NEB; Frankfurt, Germany).

Techniques: Transfection, Construct, Western Blot, Control, Expressing

Journal: Frontiers in immunology

Article Title: Detrimental NFKB1 missense variants affecting the Rel-homology domain of p105/p50.

doi: 10.3389/fimmu.2022.965326

Figure Lengend Snippet: FIGURE 4 Impaired p50-mediated DNA-binding activity uncovers deleterious NFKB1 mutations and loss-of-function variants. HEK293T cells were transiently transfected with the indicated p105 or p50 variants. NF-kB DNA-binding activities were determined by EMSA using nuclear extracts. (A) In cells transfected with p105 expression constructs, nuclear DNA-binding activity originates from cell-intrinsic generation of p50 by processing of precursor proteins. Reduced or absent DNA-binding denotes decaying and DNA-binding deficient variants or might indicate subtle defects e.g. related to processing or nuclear transfer. Please note the consumption of the free probe when using overexpressed p50 as positive control. Representative results of three independent experiments are shown. Electrophoresis was carried out twice. (B) Immediate expression of p50 (i.e. not generated via precursor processing) indicates deleterious p50 defects and DNA-binding-deficiency. Please note the reduced size of the shifted band with the R192W variant. Representative results of five independent experiments are shown.

Article Snippet: Primary antibodies were rabbit-anti-NF-kB1 #13586 (raised against residues surrounding Ile415 of mouse NF-kB1 to simultaneously detect p105 and p50) and mouseanti-beta-actin #3700 (both from Cell Signaling; NEB; Frankfurt, Germany).

Techniques: Binding Assay, Activity Assay, Transfection, Expressing, Construct, Positive Control, Electrophoresis, Generated, Variant Assay

Journal: Frontiers in immunology

Article Title: Detrimental NFKB1 missense variants affecting the Rel-homology domain of p105/p50.

doi: 10.3389/fimmu.2022.965326

Figure Lengend Snippet: FIGURE 5 The inhibitory effect on RelA-dependent promoter activation can be augmented by co-expression of mutant p105 but can either be attenuated or intensified by mutant p50. HEK293T cells were transiently transfected with a synthetic reporter gene construct (100ng), composed of an NF- kB responsive promoter driving the expression of the red fluorescent protein tdTomato. Reporter expression was switched on to maximum levels by co-expression of RelA (5 to 7.5ng vector). An excess of wildtype or mutant EGFP-p105 or EGFP-p50 expression vectors (300ng each; green fluorescence) was added to inhibit the RelA-mediated reporter activation. Expression of the reporter gene and the p105- or p50 fusion constructs was monitored at 48h (not shown) and 72h after transfections by recording the red (4-8 scans per plate) and green (3-6 scans per plate) fluorescence intensities, respectively. Average fold values were calculated from three independent experiments with the baseline intensities of the “reporter only” controls set as 1-fold. Each color in the three-colored bar graphs indicates one experiment (proportional illustration, no absolute values). Microscopic images show one representative example of the three independent experiments each. The full panels with absolute reporter activity and expression values for each variant are shown in the Supplementary Figures 2-3 and Supplementary Table 2. (A) Moderate RelA-dependent reporter activity is observed with co-expression of wildtype p105. Decreased reporter expression upon co-expression of mutant p105 compared to wildtype p105 indicates protein defects and might be due to less or absent processing to p50 and/ or cytoplasmic retention of RelA by mutant p105, respectively. Average values were calculated from three independent experiments using 5ng, 6ng and 7.5ng of the RelA vector, respectively. (B) Co-expression of wildtype p50 limits the RelA-dependent reporter activation, probably due to the assembly of excess homodimeric transcriptional repressors. Defects of p50 caused by single amino acid changes are indicated by either reduced or increased reporter activation compared to wildtype p50 and might be due to less expression, loss of DNA binding activity or other defects. Average values were calculated from three independent experiments using 5ng RelA vector each.

Article Snippet: Primary antibodies were rabbit-anti-NF-kB1 #13586 (raised against residues surrounding Ile415 of mouse NF-kB1 to simultaneously detect p105 and p50) and mouseanti-beta-actin #3700 (both from Cell Signaling; NEB; Frankfurt, Germany).

Techniques: Activation Assay, Expressing, Mutagenesis, Transfection, Construct, Plasmid Preparation, Activity Assay, Variant Assay, Binding Assay

Journal: Frontiers in immunology

Article Title: Detrimental NFKB1 missense variants affecting the Rel-homology domain of p105/p50.

doi: 10.3389/fimmu.2022.965326

Figure Lengend Snippet: FIGURE 6 NFKB1 missense variants cause protein interaction defects and subcellular mis-localization. (A) Dotplot of selected p105/p50 interactors illustrates the protein interaction changes induced by single amino acid variants. The order of the NFKB1 baits is based on hierarchical clustering. (B) The heatmap shows the molecular context of NFKB1 variants based on the annotation score of the MS-microscopy system. The cluster tree (right side) indicates common activities.

Article Snippet: Primary antibodies were rabbit-anti-NF-kB1 #13586 (raised against residues surrounding Ile415 of mouse NF-kB1 to simultaneously detect p105 and p50) and mouseanti-beta-actin #3700 (both from Cell Signaling; NEB; Frankfurt, Germany).

Techniques: Microscopy