Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: Epigallocatechin-3-O-gallate decreases tumor necrosis factor-alpha-induced fractalkine expression in endothelial cells by suppressing NF-kappaB.

doi: 10.1159/000257494

Figure Lengend Snippet: Fig. 3. Immunoblot analysis of fractalkine protein expression in HUVECs after silencing NF-κB p65. HUVECs were transfected with NF-κB p65 siRNA or control siRNA (Cont siRNA) and treated with TNF-α with or without EGCG (50 µmol/L) for 6 h. Actin was used as the loading control. Densitometric analyses are presented as the relative ratio of fractalkine to actin. The relative ratio compared to the control is arbitrarily defined as 1. Note that silencing NF-κB p65 reduced TNF-α-induced fractalkine protein expression. Bars represent the mean±SD from 3 different experiments. *, p<0.05 vs. Cont siRNA only; #, p<0.05 vs. TNF-α plus Cont siRNA.

Article Snippet: Materials and Methods Materials and endothelial cell culture Anti-fractalkine antibody (full-length fractalkine: Torrey Pines BioLabs, Houston, TX, USA), anti-phospho-p65 antibody (Ser536) (Cell Signaling Technology, Beverly, MA, USA), anti-p65 antibody (Upstate Biotechnology, Lake Placid, NY, USA), recombinant human TNF-α (R&D Systems, Minneapolis, MN, USA) were used in this study.

Techniques: Western Blot, Expressing, Transfection, Control

Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: Epigallocatechin-3-O-gallate decreases tumor necrosis factor-alpha-induced fractalkine expression in endothelial cells by suppressing NF-kappaB.

doi: 10.1159/000257494

Figure Lengend Snippet: Fig. 4. EGCG suppresses TNF-α-induced IκB-α phosphorylation, degradation of IκB-α and p65 phosphorylation (A) Immunoblot analysis of phosphorylated IκB-α in total cell extract. HUVECs were pre-treated with 50 µmol/L EGCG for 30 min and stimulated with TNF- α (10 ng/mL) and EGCG (50 µmol/L) for 1 h. Control cells (CB) received vehicle alone. Total cell extracts were prepared and analyzed for phosphorylation of IκB-α. Twenty micrograms of total protein was used in each experiment. Densitometric analyses are presented as the relative ratio of phospho-IκB- α to IκB- α. Bars represent the mean±SD from 3 different experiments. *, p<0.05 vs. CB; #, p<0.05 vs. TNF-α. (B) Immunoblot analysis of IκB-α in total cell extract. HUVECs were pre-treated with 50 µmol/L EGCG for 30 min and stimulated with TNF-α (10 ng/mL) and EGCG (50 µmol/L) for 4 h. Control cells (CB) received vehicle alone. Total cell extracts were prepared and analyzed for IκB- α. Twenty micrograms of total protein was used in each experiment. Densitometric analyses are presented as the relative ratio of IκB-α to actin. Bars represent the mean±SD from 3 different experiments. *, p<0.05 vs. CB; #, p<0.05 vs. TNF-α. (C) Immunoblot analysis of phosphorylated p65 subunit of NF-κB in total cell extracts. HUVECs were pre-treated with 50 µmol/L EGCG for 30 min and stimulated with TNF-α (10 ng/mL) and EGCG (50 µmol/L) for the indicated times. Control cells (CB) received vehicle alone. Total cell extracts were prepared and analyzed for phosphorylation of NF-κB p65. Twenty micrograms of nuclear protein was used in each experiment. Densitometric analyses are presented as the relative ratio of phospho-p65 to p65 subunit of NF-κB. Bars represent the mean±SD from 3 different experiments. *, p<0.05 vs. CB; #, p<0.05 vs. TNF-α. (D) Immunoblot analysis of the p65 subunit of NF-κB in nuclear and cytoplasmic protein extracts. HUVECs were pre-treated with 50 µmol/L EGCG for 30 min and stimulated with TNF- α (10 ng/mL) and EGCG (50 µmol/L) for 4 h. Nuclear and cytoplasmic protein extracts were prepared as described in the Material and Methods sections. Twenty micrograms of nuclear protein was used in each experiment. Note that TNF-α-induced nuclear translocation of NF-κB p65 was increased by about ~4.0 fold compared to control. However, co-treatment with TNF-α (10 ng/mL) and EGCG (50 µmol/L) suppressed nuclear translocation of NF-κB p65 compared to treatment with TNF-α alone. EGCG alone had no effect on the nuclear translocation of NF-κB p65. Densitometric analyses are presented as the relative ratio of nuclear p65 to cytosolic p65 of NF-κB. Bars represent the mean±SD from 3 different experiments. *, p<0.05 vs. CB; #, p<0.05 vs. TNF-α.

Article Snippet: Materials and Methods Materials and endothelial cell culture Anti-fractalkine antibody (full-length fractalkine: Torrey Pines BioLabs, Houston, TX, USA), anti-phospho-p65 antibody (Ser536) (Cell Signaling Technology, Beverly, MA, USA), anti-p65 antibody (Upstate Biotechnology, Lake Placid, NY, USA), recombinant human TNF-α (R&D Systems, Minneapolis, MN, USA) were used in this study.

Techniques: Phospho-proteomics, Western Blot, Control, Translocation Assay

Journal: Nature communications

Article Title: PPARγ is an E3 ligase that induces the degradation of NFκB/p65.

doi: 10.1038/ncomms2270

Figure Lengend Snippet: Figure 1 | PPARg induces the degradation of p65. (a) Confocal immunofluorescence staining of PPARg in HT29 cells. Scale bar, 30 mm. (b) Cell lysates from wild-type (WT) or PPARg / MEF cells were subjected to western blotting as shown. (c) HT29 cells were transfected with scrambled shRNA or PPARg shRNA for 36 h and cell lysates were subjected to western blotting. (d) WT or PPARg / MEF cells were treated with cycloheximide (CHX, 30 mg ml 1) for 0, 0.5, 1 and 2 h to inhibit de novo protein synthesis and harvested for western blotting. The levels of p65 at time 0 was set as 100% and the per cent p65 protein remaining following CHX treatment at each time point was calculated accordingly. (e) Control or PPARg-silenced HT29 cells were treated with CHX (30 mg ml 1) for 0, 1, 3 and 5 h to inhibit de novo protein synthesis and harvested for p65 western blotting. The per cent p65 protein remaining at each time point was calculated accordingly. (f,g) HEK293T cells were transfected with p65, HA-PPARg or vector control for 36 h. Subcellular fractionations were subjected to western blotting (f) or RT-PCR analysis (g). (h) HEK293T cells were transfected with p65, HA-PPARg or vector control for 36 h. Cells were treated with or without 10 mM MG132 for 6 h before cell lysis and subjected to western blotting as shown. (i) HEK293T cells were transfected with p65, HA-PPARg or vector control. After 36 h, cells were treated with CHX (30 mg ml 1) for 0, 2, 4 and 8 h to inhibit de novo protein synthesis and harvested for western blotting. The per cent p65 protein remaining at each time point was calculated accordingly. Data are triplicates from three independent experiments. DAPI, 40,6-diamidino-2-phenylindole.

Article Snippet: Ubiquitin (P4D1), PPARa, PPARb and p65 were from Santa Cruz. p52, c-Rel and RelB were from Cell Signaling Technology.

Techniques: Staining, Western Blot, Transfection, shRNA, Control, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Lysis

Journal: Nature communications

Article Title: PPARγ is an E3 ligase that induces the degradation of NFκB/p65.

doi: 10.1038/ncomms2270

Figure Lengend Snippet: Figure 2 | PPARg binds p65. (a) Confocal analysis of PPARg interaction with p65 in HT29 cells. Arrow heads show PPARg co-localized with p65. Scale bar, 30 mm. (b) HT29 or MEF cell lysates were subjected to immunoprecipitation and western blot analysis as shown. (c) HEK293Tcells were transfected with GST-p65 and HA-PPARg and subcellular fractions were subjected to GST pull-down (GST-PD) and western blotting. To inhibit protein degradation, cells were treated with 10 mM MG132 for 6 h before cell lysis. (d) Constructs of p65 (upper panel). Cell lysates of HA-PPARg, GST-RHD or GST-307-551 transfected in HEK293T cells were subjected to GST pull-down and western blotting. Cells were treated with 10 mM MG132 for 6 h before cell lysis. (e) Constructs of PPARg (upper panel). Cell lysates of p65, GST-PPARg or GST-PPARg-DLBD transfected in HEK293T cells were subjected to GST pull- down and western blotting. Cells were treated with 10 mM MG132 for 6 h before cell lysis. Data are triplicates from three independent experiments. DAPI, 40,6-diamidino-2-phenylindole.

Article Snippet: Ubiquitin (P4D1), PPARa, PPARb and p65 were from Santa Cruz. p52, c-Rel and RelB were from Cell Signaling Technology.

Techniques: Immunoprecipitation, Western Blot, Transfection, Lysis, Construct

Journal: Nature communications

Article Title: PPARγ is an E3 ligase that induces the degradation of NFκB/p65.

doi: 10.1038/ncomms2270

Figure Lengend Snippet: Figure 3 | PPARg functions as an E3 ligase to induce the ubiquitination of p65. (a) HEK293Tcells were transiently transfected with PPARg for 36 h. Cell lysates were subjected to denatured immunoprecipitation and western blotting. Cells were treated with 10 mM MG132 for 6 h before cell lysis. (b) HT29 cells were transfected with scrambled shRNA or PPARg shRNA for 36 h. Cell lysates were then subjected to denatured immunoprecipitation western blotting. Cells were treated with 10 mM MG132 for 6 h before cell lysis. (c) PPARg / MEF cells or PPARg / MEF cells were transiently transfected with PPARg, C139A or C193A separately. Cell lysates were subjected to denatured immunoprecipitation and western blotting. Cells were treated with 10 mM MG132 for 6 h before cell lysis. (d) In vitro polyubiquitin formation analysis was performed (see experimental procedures) in reaction buffer contained different E2 (UBCH3, UBCH5a, b, c) with 10ng PPARg or (e) containing UBCH3 with 10 ng PPARg, C139A or C193A as indicated. Reactions were incubated at 30 1C for 2 h. The ubiquitinated products were detected with an ubiquitin antibody. (f) GST pull-down assay in which GST-PPARg or GSTalone bound to glutathione-agarose beads was incubated with recombinant p65. (g) In vitro ubiquitination of p65 analysis was performed (see experimental procedures) in the reaction buffer contained UBCH3, p65 (10 mg) and 10 ng PPARg (WT or C139A) as indicated. Reactions were incubated at 30 1C for 2 h, resolved by SDS–PAGE and the ubiquitinated products were detected with p65 antibody. (h) HEK293 cells were transfected with PPARg, his-p65 or K28R. Cell lysates were subjected to denatured Ni-NTA pull-down and western blotted. Cells were treated with 10 mM MG132 for 6 h before cell lysis. (i) HT29 cells were transfected with his-p65 or his-p65/K28R plasmids and after 36 h, cells were treated with cycloheximide (30 mg ml 1) for 0, 0.5, 1, 2 and 5 h to inhibit de novo protein synthesis and harvested for p65 by western blot. Data are triplicates from three independent experiments.

Article Snippet: Ubiquitin (P4D1), PPARa, PPARb and p65 were from Santa Cruz. p52, c-Rel and RelB were from Cell Signaling Technology.

Techniques: Ubiquitin Proteomics, Transfection, Immunoprecipitation, Western Blot, Lysis, shRNA, In Vitro, Incubation, Pull Down Assay, Recombinant, SDS Page

Journal: Nature communications

Article Title: PPARγ is an E3 ligase that induces the degradation of NFκB/p65.

doi: 10.1038/ncomms2270

Figure Lengend Snippet: Figure 4 | Lys28 is required for PPARg-mediated p65 degradation. (a) HEK293T cells were transfected with GST-p65 and PPARg, and cell lysates were subjected to GST pull-down and SDS–PAGE. The Coomassie-stained proteins were excised and subjected to LC/MS/MS analysis. One of seven lysine residues of ubiquitin produces an isopeptide linkage with the C-terminus of another ubiqutin moiety, forming ubiquitin chains of various lengths and shapes (Lys 6, Lys 11, Lys 27, Lys 29, Lys 33 and Lys 63), but trypsin cannot cleave Gly–Gly modified lysine, therefore, ubiquitinated peptides are identified by a 114.1 Da diglycine (GG) tag on lysine residues, which is derived from the C-terminus of ubiquitin by trypsin cleavage. The full tryptic peptide LIFAGK*QLEDGR with K48 modified by GG is shown. K* depicts the lysine residue modified by isopeptide linkages. (b) HEK293T cells were transfected with HA-PPARg, HA-ubiquitin (Ub) or HA-ubiquitin mutant (K0 or K48R) plasmids. Cell lysates were subjected to denatured immunoprecipitation and western blotting. Cells were treated with 10 mM MG132 for 6 h before cell lysis. (c) A schematic representation of the lysine mutants of p65/RHD used in this study (upper panel). (d) HEK293Tcells were transfected with HA-PPARg, GST-p65 or with the K-R mutant plasmids. Cell lysates were subjected to western blotting. (e) NFkB promoter activity in HA-PPARg, p65 or the K-R mutants transfected in HEK293Tcells. Results are expressed as means±s.e.m. (n ¼ 3).

Article Snippet: Ubiquitin (P4D1), PPARa, PPARb and p65 were from Santa Cruz. p52, c-Rel and RelB were from Cell Signaling Technology.

Techniques: Transfection, SDS Page, Staining, Liquid Chromatography with Mass Spectroscopy, Ubiquitin Proteomics, Derivative Assay, Residue, Mutagenesis, Immunoprecipitation, Western Blot, Lysis, Activity Assay

Journal: Nature communications

Article Title: PPARγ is an E3 ligase that induces the degradation of NFκB/p65.

doi: 10.1038/ncomms2270

Figure Lengend Snippet: Figure 5 | PPARg-induced p65 degradation is independent of its transcriptional activity. (a) HEK293 cells were transfected with Flag-PPARg or Flag-PPARg-DNLS and stained with Flag antibody and visualized by confocal microscopy. Scale bar, 30 mm. (b) HEK293T cells were transfected with PPRE3-luciferase reporter and PPARg or mutant plasmids as indicated for 36 h, and PPARg transcriptional activity were assayed. (c) HEK293T cells were transfected with the plasmids as indicated, and cell lysates were subjected to denatured Ni-NTA pull-down and western blotting. Cells were treated with 10 mM MG132 for 6 h before cell lysis. (d) Construct of PPARg and mutant site (upper panel). HEK293T cells were transfected with the plasmids as indicated and cell lysates were subjected to western blotting.

Article Snippet: Ubiquitin (P4D1), PPARa, PPARb and p65 were from Santa Cruz. p52, c-Rel and RelB were from Cell Signaling Technology.

Techniques: Activity Assay, Transfection, Staining, Confocal Microscopy, Luciferase, Mutagenesis, Western Blot, Lysis, Construct

Journal: Nature communications

Article Title: PPARγ is an E3 ligase that induces the degradation of NFκB/p65.

doi: 10.1038/ncomms2270

Figure Lengend Snippet: Figure 6 | Ligands of PPARg increases PPARg E3 activity. (a,b) HT29 cells were treated with 100 mM TROG or PIOG for 15 min and cell lysates were subjected to immunoprecipitation and western blotting as indicated. (c) HT29 cells were pretreated with TROG/PIOG for 15 min and then treated with cycloheximide (30 mg ml 1) for 0, 1, 2 and 3 h to inhibit de novo protein synthesis and harvested for western blotting. The per cent p65 protein remaining at each time point was calculated accordingly. The carrier dimethylsulphoxide (DMSO) served as a control. Data are triplicates from three independent experiments.

Article Snippet: Ubiquitin (P4D1), PPARa, PPARb and p65 were from Santa Cruz. p52, c-Rel and RelB were from Cell Signaling Technology.

Techniques: Activity Assay, Immunoprecipitation, Western Blot, Control

Journal: Nature communications

Article Title: PPARγ is an E3 ligase that induces the degradation of NFκB/p65.

doi: 10.1038/ncomms2270

Figure Lengend Snippet: Figure 7 | PPARg inhibits NFkB-mediated tumour growth. (a) Stable expressing plasmids in HT-29 cells (4 105) were injected subcutaneously in nude mice for 4 weeks, and tumour volume was measured. Results are expressed as means±s.e.m. (n ¼ 5; *Po0.05). p65 levels were determined from tumour lysates by western blotting (lower panel). (b) WTor PPARg / MEF cells (4 105) were injected subcutaneously in nude mice and after 4 weeks tumour volume was measured and results are expressed as means±s.e.m. (n ¼ 5; *Po0.05). Tumour lysates were subjected to western blotting and p65 expression was quantitated. (c–f) HEK293Tcells were transfected with the plasmids as indicated for 36 h, and Cox-2 or IL-1b gene expression were assayed by real-time PCR. *Po0.05. Results are expressed as means±s.e.m. (n ¼ 3). (g) HT-29 cells (1 106) were injected subcutaneously in nude mice and given PBS (Control, Ctl) or PIOG (20 mg kg 1 per day) for 3 weeks by oral gavage. Tumour volume was then measured and results are expressed as means±s.e.m. (n ¼ 5; *Po0.05). Tumour lysates were subjected to western blotting and p65 expression was quantitated. (h,i) Mice were given PBS or PIOG for 3 days by oral gavage and then injected intravenously with 2 mg murine TNF-a for 6 h. Colonic tissues were collected, and Cox-2 and IL-1b gene expression were assayed by real-time PCR. *Po0.05. Results are expressed as means±s.e.m. (n ¼ 5).

Article Snippet: Ubiquitin (P4D1), PPARa, PPARb and p65 were from Santa Cruz. p52, c-Rel and RelB were from Cell Signaling Technology.

Techniques: Expressing, Injection, Western Blot, Transfection, Gene Expression, Real-time Polymerase Chain Reaction, Control

Journal: Nature communications

Article Title: PPARγ is an E3 ligase that induces the degradation of NFκB/p65.

doi: 10.1038/ncomms2270

Figure Lengend Snippet: Figure 8 | A model depicting how PPARg induces the degradation of p65. Ligands or agonists of PPARg (for example, TROG, PIOG) induces PPARg binding to p65, which subsequently transfers the K48-linked polyubiquitin to p65 promoting its degradation in a proteasome-dependent manner and abrogated NFkB/p65-mediated inflammation and cancer.

Article Snippet: Ubiquitin (P4D1), PPARa, PPARb and p65 were from Santa Cruz. p52, c-Rel and RelB were from Cell Signaling Technology.

Techniques: Binding Assay

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

Article Title: Bacteroides fragilis enterotoxin upregulates intercellular adhesion molecule-1 in endothelial cells via an aldose reductase-, MAPK-, and NF-κB-dependent pathway, leading to monocyte adhesion to endothelial cells.

doi: 10.4049/jimmunol.1101226

Figure Lengend Snippet: FIGURE 3. Activation of NF-kB and IKK in ECs stimulated with BFT. A, HUVECs were treated with BFT (100 ng/ml) for the indicated periods. NF-kB DNA-binding activity was assessed by EMSA. B, Immunoblot results for concurrent IkBa, phospho–IKK-a/b, IKK-a, IKK-b, and actin expression levels in HUVECs under the same conditions are provided beneath the EMSA. The results are representative of three independent experiments. C, RAOECs were treated with BFT (100 ng/ml) for the in- dicated periods. NF-kB DNA-binding activity was assessed by EMSA. The results are representative of three independent experiments. D, Supershift assays using nuclear extracts from HUVECs treated with BFT (100 ng/ml) for 1 h were performed using Abs to p50, p52, p65, c-Rel, and Rel B. The results are representative of three independent experiments.

Article Snippet: Plasmids, transfection, and luciferase assays Small interfering RNAs (siRNA) against the p65 subunit of NF-kB and cjun were obtained from Qiagen (Valencia, CA) and Santa Cruz Biotechnology, respectively.

Techniques: Activation Assay, Binding Assay, Activity Assay, Western Blot, Expressing

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

Article Title: Bacteroides fragilis enterotoxin upregulates intercellular adhesion molecule-1 in endothelial cells via an aldose reductase-, MAPK-, and NF-κB-dependent pathway, leading to monocyte adhesion to endothelial cells.

doi: 10.4049/jimmunol.1101226

Figure Lengend Snippet: FIGURE 5. Relationship between NF-kB signaling and ICAM-1 expression in BFT-stimulated ECs. A, Left panel, The culture and transfection conditions are the same as those described in the legend for Fig. 4A. Right panel, HUVECs were transfected with NF-kB p65-specific silencing siRNA (siRNA) or NS-RNA for 48 h. The transfected cells were treated with BFT (100 ng/ml) for 24 h. Cells were stained with mAb against ICAM-1 and were analyzed using flow cytometry. The data represent the MFI 6 SEM of five independent experiments. *p , 0.05. B, RAOECs were pre- incubated with the NF-kB inhibitor MG-132 (50 mM) for 30 min and then treated with BFT (100 ng/ml) for another 9 h. Levels of ICAM-1 mRNA were analyzed by quantitative RT-PCR using standard RNA. Values are expressed as mean 6 SD (n = 3). The b-actin mRNA levels in each group remained relatively con- stant throughout the same period (∼107 transcripts/mg total RNA). C, RAOECs were preincubated with MG- 132 (50 mM) for 30 min and then stimulated with BFT (100 ng/ml) for another 24 h. Cells were stained with mAb against ICAM-1 and were analyzed using flow cytometry. The data represent the MFI 6 SEM (n = 3). *p , 0.05 compared with BFT alone.

Article Snippet: Plasmids, transfection, and luciferase assays Small interfering RNAs (siRNA) against the p65 subunit of NF-kB and cjun were obtained from Qiagen (Valencia, CA) and Santa Cruz Biotechnology, respectively.

Techniques: Expressing, Transfection, Staining, Cytometry, Incubation, Quantitative RT-PCR

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: T3SS-Independent Uptake of the Short-Trip Toxin-Related Recombinant NleC Effector of Enteropathogenic Escherichia coli Leads to NF-κB p65 Cleavage

doi: 10.3389/fcimb.2017.00119

Figure Lengend Snippet: rNleC is catalytically active but requires IL1β-stimulated cells for p65 cleavage. (A) 250 μg of rNleC variants were incubated for 30 min with HeLa cell lysates at 4°C and analyzed for p65-cleaving events by Western blotting. The closed arrowhead marks the full-length proteins, the arrow highlights the cleaved proteins. The bar graph depicts the densitometric evaluation of immunoblots of at least three independent experiments (mean ± SD) with the levels of significance (one-way ANOVA, followed by Bonferroni's multiple comparisons test) indicated (see below). FLAG-tagged rNleC, rNleC variants and rGFP were detected with an α-FLAG antibody. (B) Overlay of predicted 3D structures of NleC (blue) and NleC Δ208-257 (red) generated by Swiss-PdbViewer 4.1.0. (C) rNleC was incubated for the indicated time intervals with HeLa cell lysates (125 μg rNleC at 4°C) or whole cells (25 μg/ml rNleC at 37°C). Immunoblots for p65 were done to detect rNleC-dependent cleavage events. The bar graph shows the densitometric evaluation of immunoblots of at least three independent experiments (mean ± SD) with the levels of significance (two-way ANOVA, followed by Bonferroni's multiple comparison test) indicated (see below). (D) HeLa cells were incubated with 25 μg/ml rNleC for 1 h at 37°C and stimulated with 10 ng/ml IL1β for the indicated times. Cleaving events and stimulation were visualized with immunoblots for p65 and phosphorylated p65, respectively. Bar graph shows densitometric evaluation of immunoblots of at least three independent experiments (mean ± SD) with the levels of significance (one-way ANOVA, followed by Bonferroni's multiple comparison test) indicated (see below). (E) HeLa cells were pre-incubated with BAY 11-7085 inhibitor (10 μM) for 1 h, followed by incubation with rNleC (50 μg/ml) for 1 h at 37°C. Cells were stimulated with 10 ng/ml IL1β for 20 min at 37°C prior to lysis in radioimmunoprecipitation assay (RIPA) buffer. Cleavage events of p65 were analyzed by immunoblotting with α-p65 antibodies. Tubulin was used as loading control. The bar graph shows the densitometric evaluation of immunoblots of at least three independent experiments (mean ± SD) with the levels of significance (two-way ANOVA, followed by Bonferroni's multiple comparison test) indicated. ** p ≤ 0.01 and *** p ≤ 0.001. Gray boxes indicate bands from the same gel.

Article Snippet: The following antibodies were used in the study: NF-κB p65 D14E12 (Cell Signaling Technology, Danvers, MA, USA), phospho-NF-κB p65 (Cell Signaling Technology), FLAG M2 (Sigma-Aldrich, St. Louis, MO, USA), Rab5 (Cell Signaling Technology), Rab6 (Santa Cruz Biotechnology, Dallas, TX, USA), Rab7 (Cell Signaling Technology), CD63 H5C6 (Developmental Studies Hybridoma Bank, Iowa City, IA, USA), beta COP1 (Abcam, Cambridge, United Kingdom), GM130 (Cell Signaling Technology), alpha tubulin DM1A (Sigma-Aldrich), GAPDH FL335 (Santa Cruz), HRP-labeled goat anti rabbit (Dianova, Hamburg, Germany), HRP-labeled goat anti mouse (Dianova), Cy3-labeled goat anti rabbit (Dianova) Cy3-labeled goat anti mouse (Dianova).

Techniques: Incubation, Western Blot, Generated, Comparison, Lysis, Radio Immunoprecipitation, Control

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: T3SS-Independent Uptake of the Short-Trip Toxin-Related Recombinant NleC Effector of Enteropathogenic Escherichia coli Leads to NF-κB p65 Cleavage

doi: 10.3389/fcimb.2017.00119

Figure Lengend Snippet: NleC enters HeLa cells independent of the T3SS. (A) Trypsinzed HeLa cells were incubated with 25 μg/ml FITC-labeled proteins (rNleC and rTat NleC) or rGFP for the indicated time at 37°C. For quenching extracellular fluorescence Trypan Blue (TB) was added to the cells to a final concentration of 0.2% and fluorescence intensities were detected using flow cytometry. Line graphs are depicted as the mean of at least three independent experiments (mean ± SD) with the levels of significance (two-way ANOVA, followed by Bonferroni's multiple comparison test) compared to rGFP indicated (see below). To exclude cytotoxicity of the recombinant proteins 1 μg/ml PI was added to cells at the beginning of an experiment and its uptake, indicating cell death, was measured (right). Line graphs are depicted as the mean of at least three independent experiments (mean ± SD) with the levels of significance (two-way ANOVA, followed by Bonferroni's multiple comparison test) compared to medium control indicated (see below). (B) rNleC and FITC-rNleC were incubated for 30 min with HeLa cell lysates at 4°C and analyzed for p65-cleaving events by Western blotting. The closed arrowhead marks the full-length proteins, the arrow highlights the cleaved proteins. Cleavage of p65 was analyzed by Western blotting using α-p65 antibodies. FLAG-tagged rNleC, was detected with an α-FLAG antibody. Tubulin and GAPDH were used as loading controls. (C) Fluorescence microscopy of HeLa cells incubated with 50 μg/ml rNleC-FITC, rTat NleC-FITC (green), or rGFP for 1 h or 4 h at 37°C. PFA fixed cells were stained with Phalloidin TRITC (red) and Draq5 (blue). Fluorescence images were generated using a Zeiss LSM 510 microscope. Z-stacks of single cells were taken. Scale bar represents 10 μm. (D) The uptake of 25 μg/ml FITC-labeled proteins (rNleC, rNleC Δ183–187, rNleC Δ208–257) (Figure left) with extracellular fluorescence quenched by Trypan blue and PI uptake (Figure right) was monitored in trypsinized HeLa cells at 37°C. Line graphs are depicted as the mean of at least three independent experiments (mean ± SD) with the levels of significance (two-way ANOVA, followed by Bonferroni's multiple comparison test) compared to rGFP (left graph) or medium control (right graph) indicated (see below). * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, and **** p ≤ 0.0001

Article Snippet: The following antibodies were used in the study: NF-κB p65 D14E12 (Cell Signaling Technology, Danvers, MA, USA), phospho-NF-κB p65 (Cell Signaling Technology), FLAG M2 (Sigma-Aldrich, St. Louis, MO, USA), Rab5 (Cell Signaling Technology), Rab6 (Santa Cruz Biotechnology, Dallas, TX, USA), Rab7 (Cell Signaling Technology), CD63 H5C6 (Developmental Studies Hybridoma Bank, Iowa City, IA, USA), beta COP1 (Abcam, Cambridge, United Kingdom), GM130 (Cell Signaling Technology), alpha tubulin DM1A (Sigma-Aldrich), GAPDH FL335 (Santa Cruz), HRP-labeled goat anti rabbit (Dianova, Hamburg, Germany), HRP-labeled goat anti mouse (Dianova), Cy3-labeled goat anti rabbit (Dianova) Cy3-labeled goat anti mouse (Dianova).

Techniques: Incubation, Labeling, Fluorescence, Concentration Assay, Flow Cytometry, Comparison, Recombinant, Control, Western Blot, Microscopy, Staining, Generated

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: T3SS-Independent Uptake of the Short-Trip Toxin-Related Recombinant NleC Effector of Enteropathogenic Escherichia coli Leads to NF-κB p65 Cleavage

doi: 10.3389/fcimb.2017.00119

Figure Lengend Snippet: rNleC enters eukaryotic cells by endocytosis via lipid rafts and is subsequently routed via the endosomal-lysosomal pathway. (A) Adherent HeLa cells were pre-incubated with the indicated endocytosis inhibitors for 1 h and treated with FITC-labeled rNleC (50 μg/ml) for 4 h at 37°C. Trypsinized cells were quenched with Trypan Blue (final concentration 0.2%) and fluorescence was subsequently measured by flow cytometry. The bar graph shows the results of at least three independent experiments (mean ± SD) with the levels of significance (one-way ANOVA, followed by Bonferroni's multiple comparison test) indicated (see below). (B) Adherent HeLa cells were pre-incubated with MβCD (50 mM) for 1 h and treated with rNleC (50 μg/ml) for 4 h at 37°C. Cells were stimulated with 10 ng/ml IL1β for 20 min at 37°C. Cells were lysed in RIPA buffer for 30 min at 4°C. Cleavage of p65 was analyzed by Western blotting using α-p65 antibodies. FLAG-tagged rNleC, was detected with an α-FLAG antibody. Tubulin and GAPDH were used as loading controls. The bar graph shows the results of at least three independent experiments (mean ± SD) with the levels of significance (two-way ANOVA, followed by Bonferroni's multiple comparison test) indicated (see below). gray boxes indicate bands from the same gel. (C) Fluorescence microscopy of HeLa cells incubated with 50 μg/ml FITC-labeled rNleC for the indicated time points at 37°C. PFA-fixed cells were stained with Rab5, Rab7 and CD63 and Cy3-labeled secondary antibodies (red) to show co-localization with early endosomes (Rab5) late endosomes (Rab7), and early lysosomes (CD63). Draq5-staining (blue) was used to visualize the nucleus. Fluorescence images were generated using a confocal laser-scanning microscope (Zeiss LSM 510). Z-stacks of single cells were taken. Scale bars represent 10 μm. ** p ≤ 0.01.

Article Snippet: The following antibodies were used in the study: NF-κB p65 D14E12 (Cell Signaling Technology, Danvers, MA, USA), phospho-NF-κB p65 (Cell Signaling Technology), FLAG M2 (Sigma-Aldrich, St. Louis, MO, USA), Rab5 (Cell Signaling Technology), Rab6 (Santa Cruz Biotechnology, Dallas, TX, USA), Rab7 (Cell Signaling Technology), CD63 H5C6 (Developmental Studies Hybridoma Bank, Iowa City, IA, USA), beta COP1 (Abcam, Cambridge, United Kingdom), GM130 (Cell Signaling Technology), alpha tubulin DM1A (Sigma-Aldrich), GAPDH FL335 (Santa Cruz), HRP-labeled goat anti rabbit (Dianova, Hamburg, Germany), HRP-labeled goat anti mouse (Dianova), Cy3-labeled goat anti rabbit (Dianova) Cy3-labeled goat anti mouse (Dianova).

Techniques: Incubation, Labeling, Concentration Assay, Fluorescence, Flow Cytometry, Comparison, Western Blot, Microscopy, Staining, Generated, Laser-Scanning Microscopy

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: T3SS-Independent Uptake of the Short-Trip Toxin-Related Recombinant NleC Effector of Enteropathogenic Escherichia coli Leads to NF-κB p65 Cleavage

doi: 10.3389/fcimb.2017.00119

Figure Lengend Snippet: rNleC escapes endosomes upon structural rearrangements at low pH. (A) Fold change in TNS fluorescence of rNleC, rNleC variants, and rGFP between pH 7.5 and pH 4.0. 1.5 μM recombinant protein was incubated with 150 μM TNS for 15 min at RT. Data are presented after subtraction of background fluorescence, followed by calculation of the fold change in TNS fluorescence at 440 nm and are the mean ± SD of three independent experiments. The statistical significance (one-way ANOVA, followed by Bonferroni's multiple comparison test) compared to rNleC is indicated (see below). (B) HeLa cells were pre-incubated for 2 h with 10 nM Bafilomycin A1 followed by incubation with 25 μg/ml rNleC for 30 min at 37°C. Cells were stimulated with 10 ng/ml IL1β for 20 min at 37°C. Cells were lysed in RIPA buffer for 30 min at 4°C. Cleavage of p65 was analyzed by Western blotting using α-p65 antibodies. Tubulin was used as loading control. gray boxes indicate bands from the same gel. The bar graph shows the densitometric evaluation of immunoblots of at least three independent experiments (mean ± SD) with level of significance (two-way ANOVA, followed by Bonferroni's multiple comparison test) indicated. * p ≤ 0.05.

Article Snippet: The following antibodies were used in the study: NF-κB p65 D14E12 (Cell Signaling Technology, Danvers, MA, USA), phospho-NF-κB p65 (Cell Signaling Technology), FLAG M2 (Sigma-Aldrich, St. Louis, MO, USA), Rab5 (Cell Signaling Technology), Rab6 (Santa Cruz Biotechnology, Dallas, TX, USA), Rab7 (Cell Signaling Technology), CD63 H5C6 (Developmental Studies Hybridoma Bank, Iowa City, IA, USA), beta COP1 (Abcam, Cambridge, United Kingdom), GM130 (Cell Signaling Technology), alpha tubulin DM1A (Sigma-Aldrich), GAPDH FL335 (Santa Cruz), HRP-labeled goat anti rabbit (Dianova, Hamburg, Germany), HRP-labeled goat anti mouse (Dianova), Cy3-labeled goat anti rabbit (Dianova) Cy3-labeled goat anti mouse (Dianova).

Techniques: Fluorescence, Recombinant, Incubation, Comparison, Western Blot, Control

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: T3SS-Independent Uptake of the Short-Trip Toxin-Related Recombinant NleC Effector of Enteropathogenic Escherichia coli Leads to NF-κB p65 Cleavage

doi: 10.3389/fcimb.2017.00119

Figure Lengend Snippet: Uptake and intracellular trafficking of rNleC. (A) rNleC enters eukaryotic cells via lipid rafts and follows the endosomal-lysosomal pathway. Upon acidification of endosomes, rNleC performs a change in its three-dimensional structure and escapes from endosomal compartments into the cytosol. (EE: early endosomes, LE: late endosomes, LY: lysosomes). Protein that does not escape from endosomes is degraded. (B) Cytosolic rNleC only cleaves NF-κB p65 when the NF-κB signaling pathway is activated (for example by IL1β). Cleavage of p65 results in a C-terminal p65 fragment bound to p50, which translocates into the nucleus and an N-terminal fragment bound to RPS3, which remains in the cytosol. Without the specifier RPS3, RPS3-depent NF-κB genes such as the pro-inflammatory cytokines IL1β, IL8, or TNFα are not transcribed and innate immune responses are dampened.

Article Snippet: The following antibodies were used in the study: NF-κB p65 D14E12 (Cell Signaling Technology, Danvers, MA, USA), phospho-NF-κB p65 (Cell Signaling Technology), FLAG M2 (Sigma-Aldrich, St. Louis, MO, USA), Rab5 (Cell Signaling Technology), Rab6 (Santa Cruz Biotechnology, Dallas, TX, USA), Rab7 (Cell Signaling Technology), CD63 H5C6 (Developmental Studies Hybridoma Bank, Iowa City, IA, USA), beta COP1 (Abcam, Cambridge, United Kingdom), GM130 (Cell Signaling Technology), alpha tubulin DM1A (Sigma-Aldrich), GAPDH FL335 (Santa Cruz), HRP-labeled goat anti rabbit (Dianova, Hamburg, Germany), HRP-labeled goat anti mouse (Dianova), Cy3-labeled goat anti rabbit (Dianova) Cy3-labeled goat anti mouse (Dianova).

Techniques:

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

Article Title: DC-SCRIPT Regulates IL-10 Production in Human Dendritic Cells by Modulating NF-κBp65 Activation.

doi: 10.4049/jimmunol.1402924

Figure Lengend Snippet: FIGURE 2. TLR-induced IL-10 produc- tion in SCRIPT-KD-DCs depends on NF- kBp65. (A) Immature SCRIPT-KD-DCs and Ctrl-DCs were incubated with BAY11- 7082 for 1 h, followed by a 24-h incubation with R848. Supernatants were harvested and assayed for the effect of NF-kB inhi- bition on IL-10 production. Statistics: Stu- dent two-tailed paired t test, n = 6; error bars = SEM. (B) Immature SCRIPT-KD- DCs and Ctrl-DCs were stimulated for 1 h with R848. Nuclear lysates were isolated and allowed to react with the consensus sequence for NF-kB on a precoated 96-well plate. Different subunits of NF-kB were detected using mAbs for the protein of in- terest, together with a colorimetric readout. Statistics: one-way ANOVA with a Bonfer- roni posttest; n = 4–5; error bars = SEM. *p , 0.05, **p , 0.01.

Article Snippet: Cells were blocked and stained in PBS with 1% BSA, 0.05% NaN3, and 2% human serum, using 1:100 diluted acetyl-K310 NF-kBp65 Ab (catalog no. 3045; Cell Signaling Technology) or isotype control (rabbit IgG; Jackson Immunoresearch).

Techniques: Incubation, Two Tailed Test, Isolation, Sequencing

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

Article Title: DC-SCRIPT Regulates IL-10 Production in Human Dendritic Cells by Modulating NF-κBp65 Activation.

doi: 10.4049/jimmunol.1402924

Figure Lengend Snippet: FIGURE 3. DC-SCRIPT knockdown increases phosphorylation of NF- kBp65. SCRIPT-KD-DCs or Ctrl-DCs were stimulated with R848 for 0–120 min. Protein lysates were assayed by Western blot for phosphorylated NF-kBp65, total NF-kBp65, and actin as loading control. (A) Represen- tative donor displaying that DC-SCRIPT knockdown induces a higher phosphorylated NF-kBp65. (B and C) p-NF-kBp65 and total NF-kBp65 were quantified for a range of donors. Statistics: Ctrl-DCs were compared with SCRIPT-KD-DCs for each time point, using the Student two-tailed t test (n = 3–4). Error bars = SEM. *p , 0.05.

Article Snippet: Cells were blocked and stained in PBS with 1% BSA, 0.05% NaN3, and 2% human serum, using 1:100 diluted acetyl-K310 NF-kBp65 Ab (catalog no. 3045; Cell Signaling Technology) or isotype control (rabbit IgG; Jackson Immunoresearch).

Techniques: Knockdown, Phospho-proteomics, Western Blot, Control, Two Tailed Test

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

Article Title: DC-SCRIPT Regulates IL-10 Production in Human Dendritic Cells by Modulating NF-κBp65 Activation.

doi: 10.4049/jimmunol.1402924

Figure Lengend Snippet: FIGURE 4. DC-SCRIPT knockdown increases acetylation of NF-kBp65. Ctrl- DCs or SCRIPT-KD-DCs (SC) were stim- ulated for 0–60 min with R848, followed by intracellular acetyl NF-kBp65 (K310) FACS staining. (A and B) Overlays between unstimulated and 30-min R848-stimulated moDCs. (C) Positive control: LPS + anti– DC-SIGN Ab (DCN46). (D and E) moDCs were pretreated for 1 h with the HATi AA or vehicle control before R848 stimulation to assay the effect of HATs on the observed acetylation. The line going through (A)–(E) indicates the peak of unstimulated SCRIPT- KD-DCs. (F) Summary of the relative fluo- rescence intensity (FI) for siRNA-treated moDCs stimulated with R848. Values are normalized to the mean FI for unstimulated Ctrl-DCs. Statistics: ANOVA with a Bon- ferroni posttest, n = 3–4. *p , 0.05, **p , 0.01, ***p , 0.001.

Article Snippet: Cells were blocked and stained in PBS with 1% BSA, 0.05% NaN3, and 2% human serum, using 1:100 diluted acetyl-K310 NF-kBp65 Ab (catalog no. 3045; Cell Signaling Technology) or isotype control (rabbit IgG; Jackson Immunoresearch).

Techniques: Knockdown, Staining, Positive Control, Control

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

Article Title: DC-SCRIPT Regulates IL-10 Production in Human Dendritic Cells by Modulating NF-κBp65 Activation.

doi: 10.4049/jimmunol.1402924

Figure Lengend Snippet: FIGURE 6. DC-SCRIPT knockdown increases NF-kBp65 binding to the IL10 enhancer. SCRIPT-KD-DCs and Ctrl-DCs were stimulated for 60 min with R848 and ChIPed with an NF-kBp65 Ab or isotype control (IgG). The ChIPed product and input chromatin for the ChIP was assayed for IL10 enhancer binding by qPCR. The recovery was calculated by relating the ChIPed product to the input. Statistics: ANOVA with a Bonferroni posttest; n = 4; error bars = SEM. *p , 0.05, **p , 0.01.

Article Snippet: Cells were blocked and stained in PBS with 1% BSA, 0.05% NaN3, and 2% human serum, using 1:100 diluted acetyl-K310 NF-kBp65 Ab (catalog no. 3045; Cell Signaling Technology) or isotype control (rabbit IgG; Jackson Immunoresearch).

Techniques: Knockdown, Binding Assay, Control