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: Nature Communications

Article Title: Unconventional structure and mechanisms for membrane interaction and translocation of the NF-κB-targeting toxin AIP56

doi: 10.1038/s41467-023-43054-z

Figure Lengend Snippet: a Localization of the putative pH-sensing residues selected for replacement on the three-dimensional structure of the AIP56 catalytic domain. Cartoon (left) and surface representation of AIP56 catalytic domain with (middle) or without (right) the middle domain. The catalytic residues (H165, E166 and H169) are shown in red, the putative pH-sensing residues in orange and the D209-K247 hairpin in marine blue. The cysteine residues (pink sticks) forming the disulfide bridge (yellow) are also shown. b Analysis of NF-kB p65 cleavage in mouse bone marrow-derived macrophages (mBMDM) by V5 plus His-tagged AIP56 variants. Cleavage of p65 was assessed by western blotting (upper panel; chromogenic detection) and protein loading by staining the membranes with Ponceau S (lower panel). The result shown is representative of six ( n = 6) independent experiments. c Peak ANS (8-Anilino-1-naphthalenesulfonic acid) fluorescence measured at 475 nm for the indicated pH, normalized by subtracting the corresponding values at pH 7 (fluorescence due to conformational changes caused by the mutation and not due to acidification). The measurement curves for each pH at different wavelengths are shown in Supplementary Fig. . The results shown are representative of at least three ( n = 3) independent experiments. AIP56, black; AIP56 E214K , blue; AIP56 E218K , purple; AIP56 H222K , green; AIP56 H231K , brown; AIP56 E234K , pink; AIP56 H231K/E234K , gray; AIP56 E214K/E218K/H222K , orange; d Coomassie Blue-stained SDS-PAGE gels from limited proteolysis of AIP56 and AIP56 H231K/E234K by Proteinase K. A and B mark the bands corresponding to the catalytic and receptor-binding domains, respectively. The results shown are representative of two ( n = 2) independent experiments. e Interaction of AIP56 or AIP56 variants with black lipid bilayers. Single-channel recordings of DiPhPC/n-decane membranes after addition of the indicated proteins to one side of the black lipid bilayer at a final concentration of 14 nM. Membrane activity was induced by acidification (pH 4.8; red arrows) of the aqueous phase at the cis-side of the chamber. Each result shown is representative of at least three ( n = 3) independent measurements. Source data for ( b ), ( c ) and ( d ) are provided in the Source data file.

Article Snippet: The anti-human NF-κB p65 C-terminal domain (clone c-20) rabbit polyclonal antibody (sc-372, dilution 1:3000) was from Santa Cruz Biotechnology and the anti-V5 (R960-25, dilution 1:5000) mouse monoclonal antibody was purchased from Invitrogen.

Techniques: Derivative Assay, Western Blot, Staining, Fluorescence, Mutagenesis, SDS Page, Binding Assay, Concentration Assay, Membrane, Activity Assay

Journal: Nature Communications

Article Title: Unconventional structure and mechanisms for membrane interaction and translocation of the NF-κB-targeting toxin AIP56

doi: 10.1038/s41467-023-43054-z

Figure Lengend Snippet: a Schematic representation of chimera Bla L19-W286 AIP56 P210-N497 . Bla, β-lactamase. b FRET-based assay to access the effect of Hsp90 inhibition on Bla delivery. The cleaved/uncleaved CCF4-AM ratios were determined by quantifying the indicated number of microscopic fields per condition. Results shown represent one out of three ( n = 3) independent experiments. Statistical significance was tested by Kruskal–Wallis nonparametric test and the adjusted p values for individual comparisons were obtained by Bonferroni correction. Data are presented as mean values ± SD. Actual p values from left-to-right and upwards: p < 0.0001, p < 0.0001, p = 0.0993, p = 0.8533, p < 0.0001, p = 0.0051; ns non-significant, CCF4 Fluorescence Resonance Energy Transfer (FRET) substrate, DMAG 17-(dimethylaminoethylamino)−17-demethoxygeldanamycin (Hsp90 inhibitor), Bla β-lactamase. c Control of 17-DMAG activity by confirming its inhibitory effect on NF-kB p65 (nuclear factor kappa-light-chain-enhancer of activated B cells subunit p65) cleavage upon AIP56 intoxication of mBMDM. A representative blot of three ( n = 3) independent experiments is shown. Loading correction was achieved by dividing the density of p65 by the respective density of the Ponceau S staining. Data are presented as mean values ± SD. Different symbols represent independent experiments. DMAG 17-(dimethylaminoethylamino)−17-demethoxygeldanamycin (Hsp90 inhibitor). Source data for ( b ) and ( c ) are provided in the Source data file.

Article Snippet: The anti-human NF-κB p65 C-terminal domain (clone c-20) rabbit polyclonal antibody (sc-372, dilution 1:3000) was from Santa Cruz Biotechnology and the anti-V5 (R960-25, dilution 1:5000) mouse monoclonal antibody was purchased from Invitrogen.

Techniques: Inhibition, Fluorescence, Förster Resonance Energy Transfer, Control, Activity Assay, Staining

Journal: Nature Communications

Article Title: Unconventional structure and mechanisms for membrane interaction and translocation of the NF-κB-targeting toxin AIP56

doi: 10.1038/s41467-023-43054-z

Figure Lengend Snippet: a V5 plus His-tagged AIP56 modified in the aspartate-rich motif (AIP56 D274S/D276-278S and AIP56 D274N/D276-278N ) is unable to cleave p65 in intact cells. Cleavage of p65 was assessed by western blotting and protein loading by Ponceau S staining. The result shown is representative of six ( n = 6) independent experiments. b V5 plus His-tagged AIP56 D274S/D276-278S and AIP56 D274N/D276-278N were unable to translocate across the host cell membrane in response to acidification. In all experiments, mock-treated cells were used as controls. NF-kB p65 cleavage was analyzed by western blotting. The result shown is representative of five ( n = 5) independent experiments. The plot shows the quantification of intact NF-kB p65 normalized for Ponceau S. Statistical significance was tested by one-way ANOVA and p values for the individual comparisons were calculated using Tukey’s HSD test. Data are presented as mean values ± SD. Actual p values from left-to-right and upwards for pH 7.0: p < 0.001, p = 0.91, p > 0.99, p = 0.005, p = 0.90, p = 0.81; for pH 4.5: p = 0.02, p > 0.99, p > 0.99, p > 0.99, p = 0.78, p = 0.36; ns non-significant. Open (without concanamycin A) or closed (with concanamycin A) symbols as well as color coding have been added to facilitate the reading of the experimental conditions, as specified below the graph. Samples were derived from the same experiment and the blots processed in parallel. NF-kB p65, nuclear factor kappa-light-chain-enhancer of activated B cells subunit p65; ConcA, concanamycin A (black); AIP56, purple, AIP56 D274S/D276-278S , red; AIP56 D274N/D276-278N , green. c AIP56 D274S/D276-278S and AIP56 D274N/D276-278N retained the ability to interact with black lipid bilayers. Proteins were used at a final concentration of 14 nM. Membrane activity was induced by acidification (pH 4.8; red arrows) of the aqueous phase at the cis-side of the chamber. Each result shown is representative of at least three ( n = 3) independent measurements. Source data for ( b ) and ( c ) are provided in the Source data file.

Article Snippet: The anti-human NF-κB p65 C-terminal domain (clone c-20) rabbit polyclonal antibody (sc-372, dilution 1:3000) was from Santa Cruz Biotechnology and the anti-V5 (R960-25, dilution 1:5000) mouse monoclonal antibody was purchased from Invitrogen.

Techniques: Modification, Western Blot, Staining, Membrane, Derivative Assay, Concentration Assay, Activity Assay

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: Pediatric research

Article Title: Perfluorocarbons decrease Chlamydophila pneumoniae-mediated inflammatory responses of rat type II pneumocytes in vitro.

doi: 10.1203/01.pdr.0000233033.82664.91

Figure Lengend Snippet: Figure 3. PF5080 reduce C. pneumoniae–mediated NF-B activation. (A and B) Representative CLSM images of untreated (control) and C. pneumoniae–incubated (30 min) ATII cells without and with PF5080 pretreatment. IB and p65 (red), F-actin cytoskeleton (green), and nuclear DNA (blue); areas of blue-red overlap are pink; the bar equals 10 m. Cytoplasmatic IB and nuclear NF-B p65 were quantified on confocal images. Shown are mean SD in arbitrary units (AU) of n 3 experiments, asterisk indicates p 0.0001 for IB in cytoplasm and p 0.0001 for p65 in nucleus. (C and D) Cytoplasmatic IB and nuclear p65 were quantified by Western blot. Shown are mean SD in arbitrary units (AU) of n 3 experiments, asterisk indicates p 0.0001 for IB in cytoplasm and p 0.0001 for p65 in nucleus.

Article Snippet: Mouse IgG anti-cHSP60 (ALX-804-072-R100) antibodies (from ALEXIS Biochemicals, AXXORA GmbH, Grünberg, Germany) and rabbit IgG anti-TNFR1 (H-271) sc-7895, goat IgG anti-TLR4 (L-14) sc-16240, mouse monoclonal IgG anti-I B (H-4) sc-1643, and rabbit IgG anti-NF- B p65 (C-20) sc-372 antibodies (from Santa Cruz Biotechnology, Heidelberg, Germany) were used for primary detection.

Techniques: Activation Assay, Control, Incubation, Western Blot

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

Journal: Science signaling

Article Title: Single-molecule imaging reveals the oligomeric state of functional TNFα-induced plasma membrane TNFR1 clusters in cells.

doi: 10.1126/scisignal.aax5647

Figure Lengend Snippet: Fig. 1. Stable reconstitution of TNFR1/2−/− MEFs with TNFR1-mEos2 rescues TNF-dependent and TNFR-mediated NF-ĸB signaling. (A) Cell lysates of TNFR1/2+/+ and TNFR1/2−/− MEFs alone or reconstituted with TNFR1-mEos2 were analyzed by Western blotting with antibodies against TNFR1 and vinculin as a loading control. Asterisks indicate nonspecific bands. HeLa cell extracts were analyzed as positive controls. Western blots are representative of three independent experiments. (B) TNF-induced TNFR1 activation at the indicated times in the indicated MEFs was evaluated by Western blotting analysis of the phosphorylation and degradation of IĸB. Vinculin was used as a loading control. Western blots are representative of three independent experiments. (C) TNF-induced nuclear translocation of p65 in the indicated MEFs was analyzed by immunofluorescence staining of p65. DNA was counterstained by DAPI. Right: Panels show the mEos2 fluorescence signal. Scale bars, 10 m. Images are representative of three independent ex- periments. (D) Evaluation of TNF-induced programmed cell death. The indicated MEFs were left untreated (−) or were treated with TNF (10 ng/ml) (T) and 20 M zVAD.fmk (Z) for 48 hours after 30-min pretreatment with 10 M BV6 (B). The percentage of propidium iodide (PI)–positive cells of the total cell number was quantified as a measure for cell death. EV, empty vector. Data are means ± SD of at least three experiments performed in triplicate. ***P < 0.001.

Article Snippet: Antibodies used were monoclonal mouse anti-human vinculin (V9131, Sigma-Aldrich), anti-human IĸB (#9242, Cell Signaling Technology), anti–phospho-IĸB (Ser32, 14D4, 2859, Cell Signaling Technology), anti–NF-B p65 (C-20, sc-372, Santa Cruz Biotechnology), mouse anti-TNFR1 H-5 (sc-8436, Santa Cruz Biotechnology), and rabbit anti-TNFR1 C25C1 (#3736S, Cell Signaling Technology).

Techniques: Western Blot, Control, Activation Assay, Phospho-proteomics, Translocation Assay, Immunofluorescence, Staining, Fluorescence, Plasmid Preparation

Journal: Virology

Article Title: Cowpox virus and other members of the orthopoxvirus genus interfere with the regulation of NF-kappaB activation.

doi: 10.1006/viro.2001.1090

Figure Lengend Snippet: FIG. 3. Cowpox virus inhibits TNF-induced nuclear accumulation of NF-kB capable of binding to kB-site oligonucleotides. Nuclear extracts were prepared both from uninfected HEK 293 cells and from cowpox virus-infected HEK 293 cells (5 PFU/cell) at various times after infection, either with or without TNF treatment (5 ng/ml for 15 min). The nuclear extracts from the cells were incubated with radiolabeled oligonucleotides containing a consensus NF-kB binding-site sequence. Resultant complex formation was analyzed by electrophoretic mobility shift assay (EMSA). (A) TNF treatment induces p50/p65 NF-kB complex formation in uninfected cells. For supershift analysis, prior to being incubated with the oligonucleotides, the extracts were preincubated with goat antibodies against the p50 (lane 3) or p65 (lane 4) subunits of human NF-kB or with normal goat serum (NGS). (B) Uninfected HEK 293 cells (lanes 1–2) or HEK 293 cells infected with cowpox virus (lanes 3–12) were harvested at the indicated hours postinfection; cells were either untreated or treated with TNF for the 15 min prior to harvest.

Article Snippet: Rabbit polyclonal anti-IkBa IgG (C-21), goat polyclonal anti-NF-kB p50 (NLS)-G IgG, and goat polyclonal anti-NF-kB p65 (C-20)-G IgG were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Techniques: Virus, Binding Assay, Infection, Incubation, Sequencing, Electrophoretic Mobility Shift Assay

Journal: International immunopharmacology

Article Title: Modulation of T lymphocyte and eosinophil functions in vitro by natural tetranortriterpenoids isolated from Carapa guianensis Aublet.

doi: 10.1016/j.intimp.2010.09.010

Figure Lengend Snippet: Fig. 6. Effect of TNTPs on subcellular localization of NFκB (p65) after OVA stimulation. NFκB protein levels were determined in nuclear extracts of splenocytes from previously sensitized C57BL/6 mice. Splenocytes (105 cells/well) were pre-treated with pooled TNTPs, TNTP1, TNTP2, TNTP3, TNTP4, TNTP5 (50 μg/ml) or dexamethasone (Dexa; 1.95 pg/ml) and stimulated with OVA (10 μg/well) for 24 h at 37 °C and 5% CO2. A total of 25 μg of protein was applied per lane (A). Densitometric analysis is shown (B). Results are expressed as the mean±SEM from triplicate wells per group, as statistically analyzed by means of analysis of variance (ANOVA), followed by Newman–Keuls– Student test or Student t test. * indicates statistically significant differences (p≤0.05) between non-stimulated and stimulated animals, whereas + indicates significant differences between stimulated and treated groups. These results are representative of three separate experiments performed with cells obtained from different mice.

Article Snippet: Rabbit anti-mouse NFkB/p65 (C-20), biotinylated goat anti-rabbit IgG were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) and streptavidin-conjugated horseradish peroxidase were purchased from Caltag Laboratories (Burlingame, CA).

Techniques: