Journal: PLoS ONE

Article Title: Participation of the Cell Polarity Protein PALS1 to T-Cell Receptor-Mediated NF-κB Activation

doi: 10.1371/journal.pone.0018159

Figure Lengend Snippet: A, Jurkat cells were transfected with nonspecific ( NS) - and PALS1 -siRNA, and left three days prior stimulation with 1 µg.ml −1 anti-CD3 and anti-CD28 for 0, 10, 20, and 30 min. Cell lysates were prepared and immunoblots were performed as indicated. GAPDH, and p65 served as loading controls. Five other experiments provided same results. B, NS - and PALS1 -siRNA transfected Jurkat cells were loaded with the calcium-sensitive dye Fluo-4 and stimulated with 1 µg.ml −1 anti-CD3 (closed symbol), or with 1 µg.ml −1 ionomycin (open symbol). Shown is the mean ± s.d. of triplicate measurements (one out of two independent experiments). C, D, Jurkat lymphocytes were transfected with NS - or with three individual siRNA sequences targeting PALS1. After three days, cells were co-transfected with siRNA and with NF-AT or NF-κB firefly luciferase reporter gene together with a control Renilla plasmid for an additional 24 hours. Cells were then stimulated with 20 or 40 ng.ml -1 PMA and 300 ng.ml −1 ionomycin (P/I), or 0.5 µg.ml −1 anti-CD3 and anti-CD28. Histograms represent the mean ± s.d. of triplicate experiments. RLU, relative light units. The inset immunoblot shows the level of PALS1 knockdown. Data shown are representative of at least five independent experiments.

Article Snippet: Antibodies to BCL10 (A-6), IkBα (C-21), MALT1 (B-12), Tubulin (TU-02), PALS1 (H-250), SCRIB (C-6), PAR6 (G-9) and p65 (C-20) were purchased from Santa Cruz.

Techniques: Transfection, Western Blot, Luciferase, Control, Plasmid Preparation, Knockdown

Journal: Scientific reports

Article Title: Polyphenols journey through blood-brain barrier towards neuronal protection.

doi: 10.1038/s41598-017-11512-6

Figure Lengend Snippet: Figure 4. Effects on neuroinflammation by Cat-sulf and Pyr-sulf. Pro-inflammatory markers were evaluated, namely (a) TNF-α release, (b) intracellular superoxide production, (c) nitric oxide, and (d) CD40 quantified in N9 microglial cells. Cells were pre-incubated for 6 h with each of the bioavailable (poly)phenol metabolite and then challenged with 300ng/mL of LPS. Statistical differences are denoted as ***p < 0.001, **p < 0.01 and *p < 0.05 relatively to lesion (LPS). (e) Microglial NF-κB p65 translocation into the nucleus after 60 minutes of LPS stimulation. Cells were pre-treated with Cat-sulf or Pyr-sulf for 6 h before LPS-stimulation. NF-κB (red); Nuclei (blue) stained with DAPI. Each capture is representative of at least 3 independent biological replicates. Scale bar: 10 µm. (f–i) Microglial NF-κB p65 phosphorylation ratio and IκBα fold change in protein levels. (f) IkBα protein levels along time after LPS stimulation and (g) after 60 min of LPS stimulation with representative western blots. (h) NF-κB activation profile along time after LPS stimulation looking at NF-κB p65 phosphorylation (ser536) ratio along time after LPS stimulation and (i)after 60 min of LPS stimulation with representative western blots. Cells were pre-treated either with Pyr-sulf or Cat-sulf before LPS stimulation. Control cells (white triangles, solid line), LPS-stimulated cells (black triangles, solid line), cells treated with Cat-sulf prior to LPS stimulation (black circles, dashed line), cells treated with Pyr-sulf prior to LPS stimulation (black squares, dotted line). Statistical differences are denoted as *p < 0.05 and **p < 0.01 relatively to lesion (LPS). Western blots were analyzed under the same experimental conditions. Data are presented as the means ± SD, n = 3.

Article Snippet: Briefly, HBMEC coverslips were incubated overnight at 4 °C with primary antibodies anti-P-gp (1:50, Calbiochem), anti-MRP1 (1:100, Millipore) and anti-BCRP (1:100, Millipore) and N9 cells coverslips were incubated overnight at 4 °C with rabbit polyclonal anti-NF-κB p65 (C-20) (1:200, Santa Cruz Biotechnology).

Techniques: Incubation, Translocation Assay, Staining, Phospho-proteomics, Western Blot, Activation Assay, Control

Journal: Basic & clinical pharmacology & toxicology

Article Title: Effects of insulin-like growth factor-1 on rotenone-induced apoptosis in human lymphocyte cells.

doi: 10.1111/j.1742-7843.2009.00472.x

Figure Lengend Snippet: Fig. 4. Rotenone (ROT) induced simultaneous NF-jB, p53 and c-Jun transcription factor activation in lymphocytes. Peripheral blood lymphocytes cells were left untreated (A–C), exposed to 250 lM rotenone (D–F), 250 nM IGF-1 (G–I), and to 250 nM IGF-1 + 250 lM rotenone (J–L) for 24 hr. After the incubation period, cells were stained with anti-NF-jB-p65 (A,D,G,J), anti-p53 (B,E,H,K) and anti-c-Jun (C,F,I,L) antibodies according to the procedure described in Materials and Methods. Notice that NF-jB, p53 and c-Jun positive-nuclei (dark brown) reflect their nuclear translocation ⁄ activation. Magnification 400· (A–L).

Article Snippet: The supplier’s protocol (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA; goat ABC staining System: cat # sc-2023) was followed for the immunocytochemistry using primary goat polyclonal antibodies NF-jB p65 (C-20)-G (Santa Cruz 2009 The Authors Journal compilation 2009 Nordic Pharmacological Society.

Techniques: Activation Assay, Incubation, Staining, Translocation Assay

Journal: PLoS ONE

Article Title: How the Venom from the Ectoparasitoid Wasp Nasonia vitripennis Exhibits Anti-Inflammatory Properties on Mammalian Cell Lines

doi: 10.1371/journal.pone.0096825

Figure Lengend Snippet: A. Western blot analysis of nuclear extracts from Raw264.7. After pre-incubation with N. vitripennis venom (5 µg/ml) for 15 minutes, cells were induced for the indicated times with LPS (1 µg/ml). Subsequently, nuclear extracts were subjected to Western blot analysis to determine p65 levels. Separation of nuclear and cytoplasmic fractions was verified using PARP as control for the nuclear fractions. B. After 15 minutes pre-incubation with N. vitripennis venom, cells were induced for the indicated times with LPS (1 µg/ml). Immunofluorescence staining was performed to visualize the trafficking of the p65 subunit. Representative results from three separate experiments are shown.

Article Snippet: Antibodies to rabbit IκBα (C-21), mouse A20 (A-12), rabbit PARP-1/2 (H-250 and rabbit NF-κB p65 (C-20) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Western Blot, Incubation, Control, Immunofluorescence, Staining

Journal: PLoS ONE

Article Title: How the Venom from the Ectoparasitoid Wasp Nasonia vitripennis Exhibits Anti-Inflammatory Properties on Mammalian Cell Lines

doi: 10.1371/journal.pone.0096825

Figure Lengend Snippet: HEK293T cells were transiently transfected by the PEI method with 100(Gal)2-50.huIL6P-luc+ and with various expression plasmids, the total amount of DNA being fixed at 250 ng, i.e., pGal4 (10 ng) or pGal4-p65 (10 ng) whether or not with pGR (25 ng). 10 −6 M DEX or 5 µg/ml of venom was added 24 hours before analysis. Lysates were made and the relative luciferase activity was determined by using β-gal values as a basis for normalization. The data are expressed as the mean ±S.D. of three biological replicates.

Article Snippet: Antibodies to rabbit IκBα (C-21), mouse A20 (A-12), rabbit PARP-1/2 (H-250 and rabbit NF-κB p65 (C-20) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Transfection, Expressing, Luciferase, Activity Assay

Journal: PLoS ONE

Article Title: How the Venom from the Ectoparasitoid Wasp Nasonia vitripennis Exhibits Anti-Inflammatory Properties on Mammalian Cell Lines

doi: 10.1371/journal.pone.0096825

Figure Lengend Snippet: TNF stimulation leads to activation of the canonical NF-κB signal transduction pathway marked by IκBα degradation and translocation of the p65-p50 dimer to the nucleus. Subsequent NF-κB DNA binding enables transcription of different genes, among which the cytokine IL-6. Venom treatment leads to the inhibition of TNF induced IL-6 gene expression. In addition, TNF activates the MAPKs,JNK, ERK1/2 and p38, and pretreatment with venom results in prolonged JNK activation. The negative NF-κB regulators A20 and IκBα are indicated and both were suppressed by the venom. Glucocorticoid binding to the cytosolic Glucocorticoid Receptor (GR) results in the dissociation of chaperoning proteins, followed by GR translocation to the nucleus, where it can interfere with the activity of NF-κB. Activated GR can also directly bind to the DNA, stimulating transcription of FKBP5, MKP1 and GILZ target genes. Only the two latter genes are transcriptionally induced by the venom. The red arrows mark the various levels at which venom of N. vitripennis interferes with the represented cellular signaling pathways.

Article Snippet: Antibodies to rabbit IκBα (C-21), mouse A20 (A-12), rabbit PARP-1/2 (H-250 and rabbit NF-κB p65 (C-20) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Activation Assay, Transduction, Translocation Assay, Binding Assay, Inhibition, Gene Expression, Activity Assay, Protein-Protein interactions

Journal: The Journal of nutrition

Article Title: 3,3'-Diindolylmethane suppresses the inflammatory response to lipopolysaccharide in murine macrophages.

doi: 10.1093/jn/138.1.17

Figure Lengend Snippet: FIGURE 2 Effects of DIM on LPS-induced NF-kB signaling in RAW264.7 cells. Serum-deprived cells were treated with DIM for 18 h in DMEM containing 1% FBS. LPS was then added and incubated for another 20 min. (A) Cell lysates were subjected to western blotting with an anti-IkBa antibody. Nuclear extracts were prepared for western blotting with an anti-p65 antibody (B) and electrophoretic mobility shift assay (C). Photographs of chemiluminescent detectionof theblots (A,B) or an autoradiography of the dried gels (C), which were representative of 3 independent experiments, are shown. The relative abundance of each band was quantified and the control levels (0 mmol/L DIM) were set at 100%. The adjusted means 6 SEM, n ¼ 3 of each band is shown above each blot. (D) Cells werecotransfected with NF-kB-Luc reporter plasmid and pCMV-b-galactosidase vector and plated in 6-well plates at 2 3 105

Article Snippet: The following reagents were purchased from the indicated suppliers: DIM, LKT Laboratories; antibodies against iNOS and COX-2, BD Transduction Laboratories; anti-NF-kB p65 (C-20) antibody, Santa Cruz Biotechnology; antibodies against inhibitor of kB (IkB)a, stress-activated protein kinase/Jun-N-terminal kinase (SAPK/JNK), p-SAPK/JNK (Thr-183/Tyr-185), c-Jun, p-c-Jun (Ser-63), extracellular signal-regulated kinase (ERK)-1/2, p-ERK-1/2 (Thr-202/Tyr-204), p38 mitogen-activated protein kinase (MAPK), and p-p38 MAPK (Thr-180/ Tyr-182), Cell Signaling Technology; pNF-kB-Luc and pAP-1-Luc containing the firefly luciferase (luc) coding sequence from Photinus pyralis (21), and pCMV-b, Takara Bio. pNF-kB-Luc contains multiple copies of NF-kB consensus sequence and pAP-1-Luc contains multiple copies of the AP-1 enhancer.

Techniques: Incubation, Western Blot, Electrophoretic Mobility Shift Assay, Autoradiography, Control, Plasmid Preparation

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Variation in Genome-Wide NF-κB RELA Binding Sites upon Microbial Stimuli and Identification of a Virus Response Profile.

doi: 10.4049/jimmunol.1800246

Figure Lengend Snippet: FIGURE 1. Stimulation of epithelial cells, RELA activation, DNA binding, and gene expression. (A) Time course of RELA activation. Detroit 562 cells were stimulated for 2 h with LPS, TNF-a, Pam2CSK4, or poly I:C, and RELA activation was determined with the NF-kB p65 TF assay at 30-min interval for 2 h. Results are average luminescence readouts of two independent experiments. (B) Differential gene expression in response to different stimuli. After stimulation with LPS for 100 min, TNF-a for 70 min, Pam2CSK4 for 80 min, or poly I:C for 110 min, RNAs were isolated from Detroit 562 cells for RNA- seq analysis. Average Log2FC of biological duplicates were used for hierarchical clustering of genes DE in at least one condition. Sets of genes UP in poly I:C only or all conditions are highlighted. (C) Annotation of RELA binding sites. ChIP-seq experiment was performed in duplicates after treatment of Detroit 562 cells with LPS for 80 min, TNF-a for 50 min, Pam2CSK4 for 60 min, or poly I:C for 90 min. The number of peaks identified for each stimulus is reported on the right. Peaks were annotated; the bar chart shows the number of peaks in the different genomic features indicated. (D) UP, DOWN, or nonregulated (NO) genes after treatment with the four stimuli were assigned RELA ChIP-seq peaks located within 50 kb (Figure legend continues)

Article Snippet: Chromatin was then incubated with antiRELA Ab (NF-kB p65 [c-20] sc-372; Santa Cruz Biotechnology) bound to protein G magnetic beads (Dyanabeads protein G; Thermo Fisher Scientific) prepared by mixing 4 mg of Ab with 40 ml of beads in PBS plus 0.5% BSA for 4–6 h at room temperature.

Techniques: Activation Assay, Binding Assay, Gene Expression, Transcription Factor Assay, Isolation, RNA Sequencing, ChIP-sequencing

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Variation in Genome-Wide NF-κB RELA Binding Sites upon Microbial Stimuli and Identification of a Virus Response Profile.

doi: 10.4049/jimmunol.1800246

Figure Lengend Snippet: FIGURE 2. Comparison of RELA binding sites across stimuli. (A) Comparison of RELA binding sites location. Genomic coordinates of RELA ChIP-seq peaks called upon each stimulus were compared and considered overlapping when the maximum distance between two peak centers was ,500 bp. The Venn diagram shows the number of peaks overlapping across the four conditions. (B) Differential binding analysis. The heatmap shows K-means clustering of the ChIP-seq signal (pooled biological duplicates) across the four stimulations. DB regions were separated into six groups with the number of peaks in each group shown on the right. (C) Annotation of DB and non-DB peaks. The percentage of peaks in each genomic feature for both sets of peaks is reported. Pearson p values from x2 test between the two sets are reported. ***p # 0.001, ****p # 0.0001.

Article Snippet: Chromatin was then incubated with antiRELA Ab (NF-kB p65 [c-20] sc-372; Santa Cruz Biotechnology) bound to protein G magnetic beads (Dyanabeads protein G; Thermo Fisher Scientific) prepared by mixing 4 mg of Ab with 40 ml of beads in PBS plus 0.5% BSA for 4–6 h at room temperature.

Techniques: Comparison, Binding Assay, ChIP-sequencing

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Variation in Genome-Wide NF-κB RELA Binding Sites upon Microbial Stimuli and Identification of a Virus Response Profile.

doi: 10.4049/jimmunol.1800246

Figure Lengend Snippet: FIGURE 3. Poly I:C–increased RELA binding. (A) GO analysis of Group 5 peaks. Using Genomic Regions Enrichment of Annotation Tools, genomic regions from Group 5 peaks were associated to the single nearest gene and GO analysis was performed. The top five biological process terms are reported in this figure together with their p value. (B) Gene expression of genes associated with Group 5 peaks. DE genes in at least one condition were assigned DB RELA peaks located within 50 kb upstream to 50 kb downstream of the gene. The boxplot shows Log2FC in each condition (average across biological duplicates) of the genes assigned to Group 5 peaks. The p values from a paired Wilcoxon test against the gene expression under poly I:C stimulation are indicated. (C) Motif analysis of Group 5 peaks. Known motifs enrichment was investigated in the set of Group 5 peaks against all RELA peaks identified across stimuli. Log10 (p values) of significantly enriched motifs are reported in the bar graph. (D) De novo motif analysis on Group 5 peaks. Top unknown motif enriched in Group 5 peaks together with the best match from JASPAR database are represented. (E) Overlap with IRF ChIP-seq data. Binding sites for IRFs were extracted from the ENCODE data and overlapped with the RELA peaks from Group 5 (yellow), DB peaks from the other groups (dark gray), or non-DB peaks (light gray). The fraction of RELA peaks overlapping IRF binding sites are reported; the Pearson p values from a x2 test against the results for Group 5 peaks are indicated. *p # 0.05, **p # 0.01, ***p # 0.001, ****p # 0.0001.

Article Snippet: Chromatin was then incubated with antiRELA Ab (NF-kB p65 [c-20] sc-372; Santa Cruz Biotechnology) bound to protein G magnetic beads (Dyanabeads protein G; Thermo Fisher Scientific) prepared by mixing 4 mg of Ab with 40 ml of beads in PBS plus 0.5% BSA for 4–6 h at room temperature.

Techniques: Binding Assay, Gene Expression, ChIP-sequencing

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Variation in Genome-Wide NF-κB RELA Binding Sites upon Microbial Stimuli and Identification of a Virus Response Profile.

doi: 10.4049/jimmunol.1800246

Figure Lengend Snippet: FIGURE 4. OASL locus as an example of stimulus-specific RELA target. (A) Genome browser view of RELA ChIP-seq signal under different stimuli. A RELA binding site with higher signal under poly I:C stimulation is detected ∼20 kb upstream of the OASL gene. (B) Validation of the poly I:C–increased peak by ChIP qPCR. Primers were designed to amplify the region highlighted in (A) (yellow). The bar chart shows the enrichment of RELA binding at this location as percentage of input recovered. (C) OASL expression upon five stimuli. Expression was determined by RT-qPCR and expressed as fold changes in OASL expression after stimulation as compared with untreated cells. (D) Inhibition of RELA binding. Cells were pretreated with BAY 11-8072 or DMSO followed by poly I:C stimulation or no treatment control. Binding of RELA at the region highlighted in yellow in (A) was investigated by ChIP qPCR and shown as the average input percentage recovered from the immunoprecipitation. (E) NF-kB regulation of OASL. Cells were pretreated with BAY 11-8072 or DMSO followed by poly I:C stimulation or no treatment control, and OASL expression was measured by RT-qPCR and represented as fold change of expression over the control. Data in (B)–(E) represents the average of two independent experiments and SD as error bars.

Article Snippet: Chromatin was then incubated with antiRELA Ab (NF-kB p65 [c-20] sc-372; Santa Cruz Biotechnology) bound to protein G magnetic beads (Dyanabeads protein G; Thermo Fisher Scientific) prepared by mixing 4 mg of Ab with 40 ml of beads in PBS plus 0.5% BSA for 4–6 h at room temperature.

Techniques: ChIP-sequencing, Binding Assay, Biomarker Discovery, ChIP-qPCR, Expressing, Quantitative RT-PCR, Inhibition, Control, Immunoprecipitation

Journal: The Journal of Experimental Medicine

Article Title: The Cdx2 homeobox gene suppresses intestinal tumorigenesis through non–cell-autonomous mechanisms

doi: 10.1084/jem.20170934

Figure Lengend Snippet: NF-κB activation and iNOS induction in the cecal lesions of AhCre ERT ::Cdx2 f/f mice. Coimmunodetection of Cdx2 and β-catenin, RelA (p65 NF-κB) and β-catenin, iNOS and β-catenin, RelA and Cdx2, and RelA and Cdx2 in the normal cecal mucosa of wild-type mice and the cecal lesions of AhCre ERT ::Cdx2 f/f mice. Open and closed arrows respectively show Cdx2 -devoid and Cdx2 -expressing surface epithelium. The asterisk points to a Cdx2 -depleted gland underneath the surface epithelium. Pictures correspond to serial sections. They were obtained in four mice of each genotype from two independent crossings. Bars, 100 µm.

Article Snippet: Primary antibodies were as follows: mouse anti–β-catenin (clone 14; dilution 1:500; BD Transduction Lab), mouse anti-CD4 (50134-M08H; dilution 1:500; Sino Biological), goat anti-CD8b (M-20, sc-1144; dilution 1:500, Santa Cruz Biotechnology), rabbit anti-Cdx1 ( ; dilution 1:1,000), mouse anti-Cdx2 (CDX2-88, F/MU392A-UC; dilution 1:500; Biogenex), rabbit anti-Cdx2 (EPR2764Y, ab76541; dilution 1:10,000; Thermo Fisher Scientific), rabbit anti-Cldn18 (38-8000; dilution 1:500; Invitrogen), rat anti-FoxP3 (FJK-16s, 14-5773-80; dilution 1:500; Affymetrix eBioscience), rabbit anti-Iba1 (orb10863; dilution 1:500; Biorbyt), rabbit anti-iNOS (M-19, sc-650; dilution 1:500; Santa Cruz Biotechnology), rabbit anti-Ki67 (RM9106-S; dilution 1:500; Thermo Fisher Scientific), rabbit anti-Muc2 (H-300, sc-15334; dilution 1:1,000; Santa Cruz Biotechnology), rabbit anti-Olfm4 (D6Y5A, mouse-specific; dilution 1:500; Cell Signaling Technology), rabbit anti-Olfm4 (ab85046, human-specific; dilution 1:500; Abcam), rabbit anti-p-Erk1/2 (D11A8, mAb5683; dilution 1:500; Cell Signaling Technology), rabbit anti–p-STAT3 (ab76315; dilution 1:500; Abcam), rabbit anti-RelA (NF-κB p65; C-20, sc-372; dilution 1:500; Santa Cruz Biotechnology), rabbit anti-Sox2 (AB5603; dilution 1:500; Millipore), rabbit anti-Sox9 ( ; dilution 1:500), rabbit anti-Tff1 ( ; dilution 1:500), rabbit anti-Tff2 ( ; dilution 1:500), and rat anti-Tomato (clone 16D7; dilution 1:250; KerFast).

Techniques: Activation Assay, Expressing

Journal: Nucleic Acids Research

Article Title: A DNA-sensing–independent role of a nuclear RNA helicase, DHX9, in stimulation of NF-κB–mediated innate immunity against DNA virus infection

doi: 10.1093/nar/gky742

Figure Lengend Snippet: DHX9 is essential for induction of IL6 and type I IFN upon MHV-68 infection. (A–D) shDHX9/3T3 ( A, B ) and shDHX9/HeLa ( C, D ) cells were mock-infected or infected with MHV-68 at MOI 2 for 6 and 12 h. Total cellular RNA was extracted and subjected to RT-qPCR for IL6 (A, C) and IFN-β (B, D) mRNA levels. ( E, F ) shDHX9/HeLa and shCtrl cells were transfected with 2 × кB-luc ( E ) or IFN-β-luc ( F ) reporter plasmid (100 ng) for 20 h, then infected with MHV-68 at MOI 2 for 12 h, and harvested for luciferase assays. ( G ) shDHX9/HeLa and shCtrl cells were transfected with 2 × кB-luc (100 ng) and HA-p65 (50 ng) for 28 h and harvested for luciferase assays. The relative luciferase activity (RLA) of each sample was determined by normalizing the activity to the sample's β-galactosidase activity. The data represent the mean ± SD of triplicate assays. *** P < 0.001 versus control, ns = nonsignificant (Student's t test).

Article Snippet: The ChIP assays involved antibodies to p65 (C-20, sc-372, Santa Cruz Biotechnology), to RNAPII (N20, sc-899, Santa Cruz Biotechnology), DHX9 (ab26271, Abcam), and negative control IgG.

Techniques: Infection, Quantitative RT-PCR, Transfection, Plasmid Preparation, Luciferase, Activity Assay, Control

Journal: Nucleic Acids Research

Article Title: A DNA-sensing–independent role of a nuclear RNA helicase, DHX9, in stimulation of NF-κB–mediated innate immunity against DNA virus infection

doi: 10.1093/nar/gky742

Figure Lengend Snippet: Endogenous DHX9 interacts with NF-κB p65 in MHV-68–infected fibroblasts and epithelial cells. ( A ) HEK293T cells were transfected with FLAG-DHX9 (3 μg) and HA-p65 (3 μg) for 36 h and infected with MHV-68 at MOI 2 for 12 h. Cell lysates were subjected to immunoprecipitation (IP) with an anti-FLAG antibody and western blot analysis with an anti-HA antibody. ( B ) Subcellular fractions of mock-infected and MHV-68-infected HeLa cells at MOI 2 were prepared at the indicated time points. PARP-1 and α-tubulin were used to confirm the integrity of nuclear and cytosolic extracts, respectively. ( C ) Two representative confocal microscopy images of HeLa cells that were either mock-infected or infected with MHV-68 at MOI 2 for 12 h. The cells were fixed and stained for endogenous DHX9 (FITC-green) and p65 (Cy3-red). DAPI staining (blue) was used to visualize nuclei. (D and E) Interactions of DHX9 with NF-κB p65 and RNAPII in fibroblasts and epithelial cells during viral infection. NIH3T3 cells ( D ) and HeLa cells ( E ) were mock-infected or infected with MHV-68 at MOI 2 for 12 h. The cytosolic and the nuclear extracts from the cells were subjected to immunoprecipitation with the anti-DHX9 antibody and immunoblotted with the indicated antibodies. The input lysates represent 10% of the extracts by volume used for each immunoprecipitation. ( F ) The cells were prepared as described in (C) and stained for endogenous DHX9 (FITC-green) and RNAPII (Cy3-red). All panels show ×1000 magnification (scale bar: 20 μm).

Article Snippet: The ChIP assays involved antibodies to p65 (C-20, sc-372, Santa Cruz Biotechnology), to RNAPII (N20, sc-899, Santa Cruz Biotechnology), DHX9 (ab26271, Abcam), and negative control IgG.

Techniques: Infection, Transfection, Immunoprecipitation, Western Blot, Confocal Microscopy, Staining

Journal: Nucleic Acids Research

Article Title: A DNA-sensing–independent role of a nuclear RNA helicase, DHX9, in stimulation of NF-κB–mediated innate immunity against DNA virus infection

doi: 10.1093/nar/gky742

Figure Lengend Snippet: Endogenous DHX9 is required for RNAPII recruitment to NF-κB–dependent antiviral promoters. ( A ) shCtrl and shDHX9/HeLa cells were infected with MHV-68 at MOI 2 for 12 h. The nuclear extract was subjected to immunoprecipitation with the anti-p65 antibody and immunoblotted with the indicated antibodies. The input lysates represent 10% of nuclear extracts by volume used for each immunoprecipitation. (B–D) shCtrl/HeLa and shDHX9/HeLa cells were infected with MHV-68 at MOI 2 for 12 h. Cell lysates were prepared and analyzed by a ChIP assay with an anti-p65 or anti-RNAPII antibody. Normal rabbit IgG was included as a control for nonspecific immunoprecipitation. The amounts of precipitated DNAs were measured by qPCR using primers flanking the promoter regions of IL6 ( B ), IFN-β ( C ), and GAPDH ( D ). The data represent mean ± SD of triplicate assays.

Article Snippet: The ChIP assays involved antibodies to p65 (C-20, sc-372, Santa Cruz Biotechnology), to RNAPII (N20, sc-899, Santa Cruz Biotechnology), DHX9 (ab26271, Abcam), and negative control IgG.

Techniques: Infection, Immunoprecipitation, Control

Journal: Nucleic Acids Research

Article Title: A DNA-sensing–independent role of a nuclear RNA helicase, DHX9, in stimulation of NF-κB–mediated innate immunity against DNA virus infection

doi: 10.1093/nar/gky742

Figure Lengend Snippet: The ATPase/helicase activity of DHX9 is required for RNAPII recruitment to NF-κB–dependent antiviral promoters. Stable cells expressing 3′ UTR shDHX9 were reconstituted with FLAG-DHX9-WT (lacking the 3′ UTR), K417R, ΔDUF, ΔDUF+NTD DHX9 , or ΔDUF+NLS SV40 (18 μg) for 36 h. The transfected cells were either mock-infected or infected with MHV-68 at MOI 2 for 12 h. ( A ) Cell lysates were subjected to western blot analysis with anti-DHX9 and anti-α-tubulin antibodies to assess the expression levels. (B–D) Cell lysates were analyzed by ChIP assays involving the anti-p65 or anti-RNAPII antibody. Normal rabbit IgG was included as a control for nonspecific immunoprecipitation. The amounts of input and precipitated DNAs were measured by qPCR with primers flanking the promoter regions of IL6 ( B ), IFN-β ( C ), and ‘ GAPDH ( D ). The data represent mean ± SD of triplicate assays.

Article Snippet: The ChIP assays involved antibodies to p65 (C-20, sc-372, Santa Cruz Biotechnology), to RNAPII (N20, sc-899, Santa Cruz Biotechnology), DHX9 (ab26271, Abcam), and negative control IgG.

Techniques: Activity Assay, Expressing, Transfection, Infection, Western Blot, Control, Immunoprecipitation

Journal: Nucleic Acids Research

Article Title: A DNA-sensing–independent role of a nuclear RNA helicase, DHX9, in stimulation of NF-κB–mediated innate immunity against DNA virus infection

doi: 10.1093/nar/gky742

Figure Lengend Snippet: DHX9 is recruited to the NF-κB p65–dependent promoters during viral infection. HeLa cells were either mock-infected or infected with MHV-68 at MOI of 2 for 12 h. (A and B) Cell lysates were prepared and analyzed by ChIP assays with an anti-p65, anti-RNAPII, or anti-DHX9 antibody. Normal rabbit IgG was included as a control for nonspecific immunoprecipitation. The amounts of precipitated DNA were measured by quantitative PCR (qPCR) involving primers flanking the promoter regions of IL6 ( A ) and IFN-β ( B ). The data represent mean ± SD of triplicate assays. ( C ) The cell lysates were prepared for ChIP, first subjected to immunoprecipitation with the anti-p65 antibody and then subjected to re-ChIP with anti-RNAPII and anti-DHX9 antibodies. Genomic DNA samples sequentially enriched in the NF-κB target gene promoters ( IL6 and IFN-β promoters) were amplified by PCR and analyzed by agarose gel electrophoresis (lanes 3–6). Reciprocal re-ChIP using the anti-RNAPII or anti-DHX9 antibody was also conducted with the first antibody followed by incubation with the second antibody as indicated (lanes 7–14). Normal IgG was included as a control for nonspecific immunoprecipitation and input lanes indicate the chromatin complexes prior to immunoprecipitation. A set of representative agarose gel images is shown from two independent experiments.

Article Snippet: The ChIP assays involved antibodies to p65 (C-20, sc-372, Santa Cruz Biotechnology), to RNAPII (N20, sc-899, Santa Cruz Biotechnology), DHX9 (ab26271, Abcam), and negative control IgG.

Techniques: Infection, Control, Immunoprecipitation, Real-time Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Incubation

Journal: The Journal of investigative dermatology

Article Title: Inhibition of tumor necrosis factor-alpha stimulated NFkappaB/p65 in human keratinocytes by alpha-melanocyte stimulating hormone and adrenocorticotropic hormone peptides.

doi: 10.1046/j.1523-1747.2002.19602.x

Figure Lengend Snippet: Figure 4. Inhibition of TNF-a stimulated NF-jB activity in HaCaT keratinocytes by a-MSH. (a) EMSA of HaCaT keratinocyte nuclear ex- tracts (5 mg protein per gel track). EMSA shows a-MSH 1^13 inhibiting NFkB p50/p65 activation by TNFb. Lane 1, control (no stimulation of HaCaTcells); lane 2, a-MSH (109 M, 60 min); lane 3,TNFa (2.0 ng per ml, 60 min)+100 M excess cold native NFkB oligonucleotide; lane 4, TNFa (2.0 ng per ml, 60 min)+100 M excess cold mutant oligonucleotide; lane 5, TNFa (2.0 ng per ml, 30 min); lane 6,TNFa (2.0 ng per ml, 60 min); lane 7, TNFa (2.0 ng per ml, 30 min)+a-MSH 1^13 (109 M); lane 8, TNFa (2.0 ng per ml, 60 min)+a-MSH 1^13 (109 M). Upper arrow (p50/p65) indicates speci¢c NFkB p50/p65 complex. Lower arrow (NS) indicates nonspeci¢c binding complex. (b) Scanning densitometry of the relative size and inten- sity of the p50/p65 band of the NFkB complex (units, cm1).

Article Snippet: Cells were then incubated with the appropriate primary antibody: (a) anti-NFkB/p65 (C-20) goat polyclonal IgG antibody (sc-109, Santa Cruz Biotechnology, Santa Cruz, CA) for 1 h at room temperature and pressure (1:100 vol/vol, in PBS); (b) anti-IkB-a (C-21) rabbit polyclonal IgG antibody (sc-371) for 1 h at room temperature and pressure (1:100 vol/ vol, in PBS); (c) anti-MC-1R (N-19) goat polyclonal IgG antibody (sc6875) for 18 h at 41C (1:50 vol/vol, in blocking bu¡er/PBS); or (d) antiMC-2R (C-16) goat polyclonal IgG (sc-6876) for 18 h at 41C (1:50 vol/ vol, in blocking bu¡er/PBS).

Techniques: Inhibition, Activity Assay, Activation Assay, Control, Mutagenesis, Binding Assay

Journal: The Journal of investigative dermatology

Article Title: Inhibition of tumor necrosis factor-alpha stimulated NFkappaB/p65 in human keratinocytes by alpha-melanocyte stimulating hormone and adrenocorticotropic hormone peptides.

doi: 10.1046/j.1523-1747.2002.19602.x

Figure Lengend Snippet: Figure 5. Immuno£uorescent labeling of HaCaT keratinocytes for the p65 subunit of NFjB, demonstrating relative transcription fac- tor activity. (a, d) Control unstimulated HaCaT keratinocytes. Inactive NFkB identi¢ed by intracellular cytoplasmic localization with no nuclear immunolabeling. (b, e) TNFa (2.0 ng per ml) stimulated HaCaT keratino- cytes (60 min). Active NFkB identi¢ed by rapid visual nuclear localization (some cytoplasmic labeling evident). (c, f) TNFa (2.0 ng per ml) stimulated HaCaT keratinocytes +a-MSH 1^13 (at 109 M). Inhibited activation of NFkB identi¢ed by a marked reduction in nuclear localization compared with TNFa alone-stimulated cells. (a^c) NFkB/p65 labeled with FITC (green) alone. (d^f) Dual labeled micrographs with NFkB/p65 (FITC) + counterlabeled nuclei (propidium iodide, red). Scale bar: 100 mM.

Article Snippet: Cells were then incubated with the appropriate primary antibody: (a) anti-NFkB/p65 (C-20) goat polyclonal IgG antibody (sc-109, Santa Cruz Biotechnology, Santa Cruz, CA) for 1 h at room temperature and pressure (1:100 vol/vol, in PBS); (b) anti-IkB-a (C-21) rabbit polyclonal IgG antibody (sc-371) for 1 h at room temperature and pressure (1:100 vol/ vol, in PBS); (c) anti-MC-1R (N-19) goat polyclonal IgG antibody (sc6875) for 18 h at 41C (1:50 vol/vol, in blocking bu¡er/PBS); or (d) antiMC-2R (C-16) goat polyclonal IgG (sc-6876) for 18 h at 41C (1:50 vol/ vol, in blocking bu¡er/PBS).

Techniques: Labeling, Activity Assay, Control, Immunolabeling, Activation Assay

Journal: The Journal of investigative dermatology

Article Title: Inhibition of tumor necrosis factor-alpha stimulated NFkappaB/p65 in human keratinocytes by alpha-melanocyte stimulating hormone and adrenocorticotropic hormone peptides.

doi: 10.1046/j.1523-1747.2002.19602.x

Figure Lengend Snippet: Figure 6. Inhibition of TNF-a stimulated NF-jB activity in Kerati- nocytes by POMC peptides. (a) Extent of inhibition by MSH peptides on TNFa-stimulated activation of NFkB/p65 in human HaCaT keratino- cytes. Cells were stimulated for 15 min with a-MSH 1^13 (m), MSH 11^13 L-Lys-L-Pro-L-Val (), or MSH 11^13 L-Lys-L-Pro-D-Val (J), followed by stimulation withTNFa (2.0 ng per ml) for 60 min. E¡ectiveness of pep- tide action alone in the absence of TNFa stimulation on NFkB/p65 is shown for a-MSH 1^13 (&), MSH 11^13 L-Lys-L-Pro-L-Val (), or MSH 11^13 L-Lys-L-Pro-D-Val (+). Signi¢cance for a-MSH 1^13 (), MSH 11^13 L-Lys-L-Pro-L-Val (+), or MSH 11^13 L-Lys-L-Pro-D-Val ( n ): n//+, po0.05; nn//++, po0.01; nnn//+++, po0.001. (b) Extent of inhibition by ACTH peptides on TNFa-stimulated activation of NFkB/p65 in human HaCaT keratinocytes. Cells were stimulated for 15 min with ACTH 1^17 (’) or ACTH 1^39 (), followed by stimulation with TNFa (2.0 ng per ml) for 60 min. E¡ectiveness of peptide action alone in the absence of TNFa stimulation on NFkB/p65 is shown for ACTH 1^17 (&) or ACTH 1^39 (J). None of the peptide concentrations studied signi¢cantly inhibited TNFa-stimulated NFkB/p65 activation.

Article Snippet: Cells were then incubated with the appropriate primary antibody: (a) anti-NFkB/p65 (C-20) goat polyclonal IgG antibody (sc-109, Santa Cruz Biotechnology, Santa Cruz, CA) for 1 h at room temperature and pressure (1:100 vol/vol, in PBS); (b) anti-IkB-a (C-21) rabbit polyclonal IgG antibody (sc-371) for 1 h at room temperature and pressure (1:100 vol/ vol, in PBS); (c) anti-MC-1R (N-19) goat polyclonal IgG antibody (sc6875) for 18 h at 41C (1:50 vol/vol, in blocking bu¡er/PBS); or (d) antiMC-2R (C-16) goat polyclonal IgG (sc-6876) for 18 h at 41C (1:50 vol/ vol, in blocking bu¡er/PBS).

Techniques: Inhibition, Activity Assay, Activation Assay

Journal: The Journal of investigative dermatology

Article Title: Inhibition of tumor necrosis factor-alpha stimulated NFkappaB/p65 in human keratinocytes by alpha-melanocyte stimulating hormone and adrenocorticotropic hormone peptides.

doi: 10.1046/j.1523-1747.2002.19602.x

Figure Lengend Snippet: Figure 7. Inhibition of TNFa-stimulated NFjB/p65 activity in normal human keratinocytes by MSH/ACTH peptides. The most e¡ective MSH/ACTH peptide was used, as previously identi¢ed from data on HaCaT keratinocytes.

Article Snippet: Cells were then incubated with the appropriate primary antibody: (a) anti-NFkB/p65 (C-20) goat polyclonal IgG antibody (sc-109, Santa Cruz Biotechnology, Santa Cruz, CA) for 1 h at room temperature and pressure (1:100 vol/vol, in PBS); (b) anti-IkB-a (C-21) rabbit polyclonal IgG antibody (sc-371) for 1 h at room temperature and pressure (1:100 vol/ vol, in PBS); (c) anti-MC-1R (N-19) goat polyclonal IgG antibody (sc6875) for 18 h at 41C (1:50 vol/vol, in blocking bu¡er/PBS); or (d) antiMC-2R (C-16) goat polyclonal IgG (sc-6876) for 18 h at 41C (1:50 vol/ vol, in blocking bu¡er/PBS).

Techniques: Inhibition, Activity Assay