Journal: bioRxiv

Article Title: Distinct systemic and gut IgA responses to bacteria of the human upper gastrointestinal tract

doi: 10.1101/2025.07.01.662496

Figure Lengend Snippet: (A) Identified genera among all strains (N) isolated from duodenal biopsies of four donors as well as a panel of control reference strains. (B) Representative flow cytometry plots showing staining of a bacterial strain with purified serum IgA. (C) Reactivity of purified serum IgA of four donors against all biopsy isolates or control strains (n = 216) given as ΔMFI values determined by flow cytometry. (D) Serum IgA reactivity against strains isolated from each of the donors. Signals are given relative to the maximum ΔMFI value obtained for each IgA sample. Horizontal lines indicate medians, and difference between groups was evaluated by Friedman’s test with Dunn’s multiple comparisons correction. **p < 0.01, ***p < 0.001, ****p < 0.0001. (E) Heat map showing serum IgA reactivity of the four donors with all included strains. Each isolate (row) is labeled with its genus. (F) Distribution of IgA-reactive bacteria. For each serum sample (donor), only strains with ΔMFI values >10% of the maximum signal are displayed. Colors indicate genera as in (E). (G) Serum IgA reactivity against isolates of the genus Neisseria . The included isolates belonged to two different species or were unidentified ( Neisseria sp. ). Horizontal lines indicate means, and difference between groups was evaluated by one-way ANOVA with Holm-Sidak multiple comparisons correction. *p < 0.05.

Article Snippet: For detection of mAb reactivity with recombinant proteins, 3 μg/mL human TG2 or 2 μg/mL E. coli Lpp protein (TargetMol) was coated in PBS followed by incubation with IgA1 mAbs in various concentrations and detection as described above.

Techniques: Isolation, Control, Flow Cytometry, Staining, Purification, Labeling, Bacteria

Journal: bioRxiv

Article Title: Distinct systemic and gut IgA responses to bacteria of the human upper gastrointestinal tract

doi: 10.1101/2025.07.01.662496

Figure Lengend Snippet: (A) Heat map showing reactivity of serum IgA isolated from a single donor (CD1629) at four discrete time points. Reactivity was assessed against all bacterial strains (rows, n = 216) by flow cytometry. (B) Correlation between mean reactivity and SD across the four time points. For each strain, the coefficient of variation (CV) was calculated as SD divided by mean reactivity. (C) Grouping of strains according to variation in reactivity over time. Only strains with ΔMFI values >10% of the maximum signal in at least one time point are displayed. Colors indicate genera as in (A).

Article Snippet: For detection of mAb reactivity with recombinant proteins, 3 μg/mL human TG2 or 2 μg/mL E. coli Lpp protein (TargetMol) was coated in PBS followed by incubation with IgA1 mAbs in various concentrations and detection as described above.

Techniques: Isolation, Flow Cytometry

Journal: bioRxiv

Article Title: Distinct systemic and gut IgA responses to bacteria of the human upper gastrointestinal tract

doi: 10.1101/2025.07.01.662496

Figure Lengend Snippet: (A) Detection of total and bacteria-reactive IgA plasma cells in duodenal biopsy single-cell suspensions by ELISPOT. Images and quantification of spots are shown for two representative isolates. (B and C) Comparison of plasma cell reactivity by ELISPOT (B) and serum IgA reactivity by flow cytometry (C) against individual isolates. Duodenal biopsies and serum samples were obtained from a single donor (CD1629) at two discrete time points. (D) Detection of bacteria-specific IgA plasma cells by staining of duodenal biopsy single-cell suspension with labeled isolates in flow cytometry. (E) Flow cytometry plot showing reactivity of mAb 1629-A01 generated from a single S. parasanguinis- binding plasma cell against the strain that was used for plasma cell isolation. (F) Reactivity of mAb 1629-A01 against a selection of isolates assessed by flow cytometry. The strain that was used for isolation of 1629-A01 is indicated in bold. Error bar indicates SD based on three experiments.

Article Snippet: For detection of mAb reactivity with recombinant proteins, 3 μg/mL human TG2 or 2 μg/mL E. coli Lpp protein (TargetMol) was coated in PBS followed by incubation with IgA1 mAbs in various concentrations and detection as described above.

Techniques: Bacteria, Clinical Proteomics, Enzyme-linked Immunospot, Comparison, Flow Cytometry, Staining, Suspension, Labeling, Generated, Binding Assay, Cell Isolation, Selection, Isolation

Journal: bioRxiv

Article Title: Distinct systemic and gut IgA responses to bacteria of the human upper gastrointestinal tract

doi: 10.1101/2025.07.01.662496

Figure Lengend Snippet: (A) Examples of lineage trees showing plasma cell clonotypes spanning gut and BM. Observed sequences are colored according to isotype. Inferred sequences are indicated as smaller white circles, and predicted germline sequences are black. Circle sizes indicate number of cells with identical Ig sequence, and numbers next to edges indicate mutations (nt). (B and C) Overview of Ig isotype usage and number of cells in clonotypes spanning gut and BM in four celiac disease patients. Each clone is represented by one BM bar and one gut bar in (B). Isotype usage among all cells belonging to shared clones is compared to the total populations of BM and gut plasma cells in (C). IgA + cells that could not be assigned to a specific subclass are indicated as IGHA1/2. IgG + cells that could not be assigned to a specific subclass were excluded (in total 34 cells in BM and 1 cell in gut). (D) ELISA binding curves showing reactivity of mAb 558-6BM with LPS of different bacterial sources, purity, and after enzymatic treatment. Error bars represent range of sample duplicates. (E) Western blot showing reactivity of mAb 558-6BM with components of E. coli O55:B5 LPS before and after treatment with mutanolysin. In addition to a band at ∼10 kDa, a high-molecular weight band (indicated with black triangle) is present in the sample without mutanolysin. (F) Comparison of mAb 558-6BM reactivity against E. coli O55:B5 LPS and E. coli Lpp (peptide component) by ELISA. Error bars represent range of sample duplicates. (G) Reactivity of ten mAbs generated from clonotypes spanning gut and BM with a selection of bacterial isolates as determined by flow cytometry.

Article Snippet: For detection of mAb reactivity with recombinant proteins, 3 μg/mL human TG2 or 2 μg/mL E. coli Lpp protein (TargetMol) was coated in PBS followed by incubation with IgA1 mAbs in various concentrations and detection as described above.

Techniques: Clinical Proteomics, Sequencing, Clone Assay, Enzyme-linked Immunosorbent Assay, Binding Assay, Western Blot, High Molecular Weight, Comparison, Generated, Selection, Flow Cytometry

Journal: bioRxiv

Article Title: Distinct systemic and gut IgA responses to bacteria of the human upper gastrointestinal tract

doi: 10.1101/2025.07.01.662496

Figure Lengend Snippet: (A) Heat map showing IgA reactivity against a selection of bacterial isolates as determined by flow cytometry. Total IgA was purified from serum of healthy donors (HD), untreated celiac disease patients (UCeD) or celiac disease patients treated with a gluten-free diet for at least one year (TCeD) (B) Assessment of IgA integrity by SDS-PAGE after incubation with bacterial culture supernatants. Brain Heart Infusion (BHI) broth alone was used as negative control and supernatant of Streptococcus pneumoniae (S.pn), which is known to express IgA1 protease, was used as positive control. One isolate of Gemella haemolysans (G.hae) was found to have IgA-degrading activity. The effect was observed using purified serum IgA of six individual donors. (C) Detection of IgA heavy chain mobility shift after incubation of purified serum IgA of five donors with supernatant of two isolates of Streptococcus infantis (S.inf) used either alone or in combination.

Article Snippet: For detection of mAb reactivity with recombinant proteins, 3 μg/mL human TG2 or 2 μg/mL E. coli Lpp protein (TargetMol) was coated in PBS followed by incubation with IgA1 mAbs in various concentrations and detection as described above.

Techniques: Selection, Flow Cytometry, Purification, SDS Page, Incubation, Negative Control, Positive Control, Activity Assay, Mobility Shift

Journal: Pathogens

Article Title: Analysis of Porcine Pro- and Anti-Inflammatory Cytokine Induction by S. suis In Vivo and In Vitro

doi: 10.3390/pathogens9010040

Figure Lengend Snippet: Cytokine release by peripheral blood mononuclear cells (PBMC) is S. suis dose-dependent, and IL-6 and TNF-α secretion is limited by IL-10. ( A ) Isolated porcine PBMC were stimulated with strain 10 or the acapsular mutant strain 10cpsΔEF at a PBMC-to-bacteria ratio of 10:1 (10 5 cfu/well) or 1:1 (1 × 10 6 cfu/well) in the presence of antibiotics. Cytokines were measured in cell culture supernatants after 42 h of incubation ( n = 5). Median and individual pigs shown. ( B ) Cytokine levels in supernatants of PBMC stimulated with S. suis strains 10 ( cps2 ), 13-00283-02 ( cps7 ) or 16085/3b ( cps9 ) at the PBMC-to-bacteria ratios 10:1 and 1:1 in the presence of antibiotics. Cytokines were measured after 42 h of incubation ( n = 3). ( C ) TNF-α and IL-6 production in presence of neutralizing anti-IL-10 antibody or with an isotype control after the stimulation of porcine PBMC with S. suis strain 10 at the same PBMC-to-bacteria ratios in the presence of antibiotics. IL-10 production was determined for reference. Cytokines from supernatants were measured after 42 h of incubation ( n = 9–11). Graphs show pooled data from four independent experiments with seven to ten week old piglets. For statistical analyses, Mann Whitney test was performed (** p < 0.01).

Article Snippet: For functional studies, recombinant porcine TNF-α (10 ng/mL) or neutralizing anti-TNF-α antibody (4 µg/mL; both R&D Systems Inc., Minneapolis, MN, USA) was added.

Techniques: Isolation, Mutagenesis, Bacteria, Cell Culture, Incubation, MANN-WHITNEY

Journal: Pathogens

Article Title: Analysis of Porcine Pro- and Anti-Inflammatory Cytokine Induction by S. suis In Vivo and In Vitro

doi: 10.3390/pathogens9010040

Figure Lengend Snippet: Monocytes are the main producers of TNF-α in response to encapsulated S. suis. ( A ) Levels of TNF-α, IL-6 and IL-10 secreted by PBMC and CD14-positive monocytes after stimulation with the wt S. suis strain 10 ( n = 4–7) or the acapsular mutant strain 10cpsΔEF for 42 h in the presence of antibiotics. PBMC were cultivated at 1 × 10 6 cells/well (200 µL) and monocytes were cultivated at 5 × 10 4 cells/well. Bacteria were always used at 1 × 10 6 cfu/well, which is equivalent to a PBMC-to-bacteria ratio of 1:1. Graphs show pooled data of four independent experiments with seven to 10 week old piglets. Depending on the resulting cell yield of the monocyte separation, fractions were stimulated with S. suis strain 10 and/or 10cpsΔEF. ( B ) Density gradient-purified granulocytes (1 × 10 6 cells/well) from different donors, but pigs of the same herd ( n = 11) were stimulated under the same conditions as the PBMC and the monocytes. The cytokines were quantified from cell culture supernatant by ELISA. Graphs show data from two independent experiments with seven to 10 week old piglets. * p < 0.05; ** p < 0.01; *** p < 0.001 according to Mann Whitney test of control (medium) versus S. suis -stimulated samples.

Article Snippet: For functional studies, recombinant porcine TNF-α (10 ng/mL) or neutralizing anti-TNF-α antibody (4 µg/mL; both R&D Systems Inc., Minneapolis, MN, USA) was added.

Techniques: Mutagenesis, Bacteria, Purification, Cell Culture, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: Pathogens

Article Title: Analysis of Porcine Pro- and Anti-Inflammatory Cytokine Induction by S. suis In Vivo and In Vitro

doi: 10.3390/pathogens9010040

Figure Lengend Snippet: TNF-α does not contribute to bacterial killing in vitro, not even in the absence of S. suis -specific antibodies. ( A ) Kinetics of TNF-α induction and bacterial survival after infection of whole blood with 2 × 10 6 cfu/mL S. suis strain 10 ( n = 3). ( B ) Blood samples of piglets with low specific IgG ( n = 2–4) or blood cells reconstituted with colostrum-deprived serum (CDS, see Methods) (no specific IgG; n = 3–5) were infected with the viable S. suis strain 10 alone or additionally treated with porcine recombinant TNF-α (rTNF-α, 10 ng/mL) or neutralizing anti-TNF-α antibody (4 µg/mL). Bacterial survival was determined after 2 h. ( C ) Relative units per mL (RU/mL) of S. suis strain 10-specific IgG in sera of different groups of piglets. RU were calculated relative to a reference serum. Blood and sera were categorized based on the amount of S. suis strain 10-specific IgG: IgG-high > 60/mL RU ( n = 3); IgG-low < 60 RU/mL ( n = 4); No IgG = 0 RU/mL, using CDS with no detectable S. suis strain 10-specific IgG antibodies for the reconstitution of porcine blood cells. Graphs show data of four independent experiments with six to eight week old piglets.

Article Snippet: For functional studies, recombinant porcine TNF-α (10 ng/mL) or neutralizing anti-TNF-α antibody (4 µg/mL; both R&D Systems Inc., Minneapolis, MN, USA) was added.

Techniques: In Vitro, Infection, Recombinant

Journal:

Article Title: Monocytes Augment Bacterial Species- and Strain-Dependent Induction of Tissue Factor Activity in Bacterium-Infected Human Vascular Endothelial Cells

doi: 10.1128/IAI.69.5.2797-2807.2001

Figure Lengend Snippet: Contribution of endogenous IL-1 or TNF-α to enhanced TFA of monocyte-EC cocultures. Monolayers of 2 × 105 EC/well were incubated for 24 h with culture medium alone (no bacteria) or with ∼4 × 107 cells of the indicated bacteria, washed, and incubated for about 15 min with culture medium with (+) or without (−) 1 μg of anti-human TNF-α MAb/ml and/or 30 ng of rIL-1ra/ml. Then, these cells were cocultured for 6 h with 2 × 105 monocytes in the absence (−) or presence (+) of anti-human TNF-α MAb and/or rIL-1ra. TFA was determined as described in Materials and Methods. Values are the means ± standard deviations for eight separate experiments with EC from different donors. P values were <0.03 (∗)and <0.02 (∗∗) versus the same stimulus in the absense of rIL-1ra and anti-human TNF-α MAb (open bars). All mean values for TFA determined in the presence of both blocking compounds were not significantly different (P > 0.05; n = 8) from values measured in the presence of rIL-1ra alone.

Article Snippet: Neutralizing MAb against human tumor necrosis factor alpha (TNF-α) was purchased from R&D Systems.

Techniques: Incubation, Bacteria, Blocking Assay

Journal: mSystems

Article Title: Association of intratumoral microbiome diversity with hepatocellular carcinoma prognosis

doi: 10.1128/msystems.00765-24

Figure Lengend Snippet: Bacterial LPS and LTA are present inside HCC tissues. (a) Representative images of multiplex immunofluorescence (multiplex IF, 20×) staining with an Opal kit. The bacteria were stained with antibodies against LPS and LTA, while CD45 and CD68 were used to identify immune cells. The area circled by the green curve indicates the IRR. Scale bars, 50 µm. (b) The boxplot of bacterial LPS and LTA intensity. Nine ROIs were selected.

Article Snippet: Staining was conducted manually using primary antibodies against the following markers: CD45 (anti-CD45, eBioscience #14-0459-82), CD68 (anti-CD68, Invitrogen #MA5-12407), LPS (anti-LPS, abcam #ab35654), and LTA (anti-LTA, Santa Cruz #sc57752).

Techniques: Multiplex Assay, Immunofluorescence, Staining, Bacteria

Journal: PLOS Pathogens

Article Title: Fungi and bacteria occupy distinct spatial niches within carious dentin

doi: 10.1371/journal.ppat.1011865

Figure Lengend Snippet: Primary and Secondary Antibodies.

Article Snippet: Gram Positive Bacteria LTA (lipoteichoic acid) , Mouse/IgG1 , ThermoFisher , MA1-7402 , 1:100.

Techniques: Bacteria, Polymer

Journal: Antibiotics (Basel, Switzerland)

Article Title: In Vitro, In Vivo and In Silico Assessment of the Antimicrobial and Immunomodulatory Effects of a Water Buffalo Cathelicidin (WBCATH) in Experimental Pulmonary Tuberculosis.

doi: 10.3390/antibiotics12010075

Figure Lengend Snippet: Figure 6. Production of pro-inflammatory cytokines in the lungs of mice infected with drug sensible strain H37Rv and treated with WBCATH. (A) Lung gene expression of TNFα determined by RT-PCR in tuberculous mice after 1 and 2 months of treatment with WBCATH (grey bars) in comparison with control animals that only received the vehicle saline solution (black bars). (B) Lung gene expression of IFNγ after 1 and 2 months of therapy with WBCATH. (C) Representative micrographs of immunohistochemistry to detect TNFα and IFNγ in the lung of tuberculous mice treated or not with WBCATH, treated mice showed more IFNγ immunostained cells in lymphocytes around blood vessels and bronchial epithelium, while in the pneumonic areas there were more TNFα immunostained macrophages. (D) These observations were confirmed by the cell count and percentage of positive cells that showed significantly higher percentages in the treated mice (n = 6). Bars represent the mean and standard deviation, and at the top of the bars are noted the significance of the T student test.

Article Snippet: Polyclonal rabbit antibodies anti-IFNγ and TNFα were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Infection, Gene Expression, Reverse Transcription Polymerase Chain Reaction, Comparison, Control, Saline, Immunohistochemistry, Cell Counting, Standard Deviation

Journal: Antibiotics (Basel, Switzerland)

Article Title: In Vitro, In Vivo and In Silico Assessment of the Antimicrobial and Immunomodulatory Effects of a Water Buffalo Cathelicidin (WBCATH) in Experimental Pulmonary Tuberculosis.

doi: 10.3390/antibiotics12010075

Figure Lengend Snippet: Figure 7. Production of protective cytokines in the lungs of MDR tuberculous mice treated with WBCATH. (A) Gene expression of TNFα after 1 and 2 months of treatment with WBCATH (grey bars), in comparison with control non-treated animals (black bars) (B) Lung gene expression of IFNγ in non-treated (black bars) and after 1 and 2 months of therapy with WBCATH (grey bars). (C) Representative micrographs of the cellular cytokine detection by immunohistochemistry, there are more TNFα immunostained macrophages in the pneumonic patches and IFN-γ immunoreactive lymphocytes around blood vessels in the treated mice. (D) The percentage of immunostained cells to TNFα and IFNγ in control and treated mice. Bars represent the mean and standard deviation of six animals. The value of statistical significance obtained by the student T-test is at the top of the bars.

Article Snippet: Polyclonal rabbit antibodies anti-IFNγ and TNFα were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Gene Expression, Comparison, Control, Immunohistochemistry, Standard Deviation

Journal: Antibiotics (Basel, Switzerland)

Article Title: In Vitro, In Vivo and In Silico Assessment of the Antimicrobial and Immunomodulatory Effects of a Water Buffalo Cathelicidin (WBCATH) in Experimental Pulmonary Tuberculosis.

doi: 10.3390/antibiotics12010075

Figure Lengend Snippet: Figure 11. Effect of antimicrobial peptide WBCATH on Mtb. (1) In the present study, we evaluated the effect of WBCATH on Mtb. (2) Two strains of Mtb were used, the drug sensible mycobacteria H37Rv and MDR mycobacteria. (3) WBCATH induced direct damage in the mycobacteria, induced the direct killing of the bacteria, and increased the macrophage’s clearance of intracellular mycobacteria. (4) Furthermore, when we evaluated the treatment with WBCATH in a murine model of advanced TB, the cathelicidin used had an effective activity in reducing the bacterial load and pneumonia by direct antimicrobial action and by promoting the expression of TNFα, IFNγ and IL-12. The treatments presented a synergistic effect with first-line anti-TB drugs. (5) All these results show that WBCATH is an effective treatment for lung disease in advanced TB in the murine model.

Article Snippet: Polyclonal rabbit antibodies anti-IFNγ and TNFα were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Bacteria, Activity Assay, Expressing

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Nonproteolytic ubiquitination regulates chromatin occupancy by the NCoR/SMRT/HDAC3 corepressor complex in MCF-7 breast cancer cells.

doi: 10.1073/pnas.2502805122

Figure Lengend Snippet: Fig. 5. (A) Western blotting with anti-TRAF6 Ab to visualize the nuclear translocation of TRAF6 in MCF-7 cells treated with IL-1β for 5 min. WBs for HDAC2 and β-tubulin were run on the cytoplasmic and nuclear extracts as a control. (B) TRAF6 interaction with NCoR in IL-1β-treated MCF7 cells by immunoprecipitation with anti-NCoR Ab. (C) Graphic representation of the NCoR protein showing the putative TRAF6 interaction motif, and sequence alignment of similar motifs found in human RIP2, IRAK2, and NCoR across species. (D) Interaction between TRAF6 and the identified interaction motif in HEK293T cells transfected with Myc-TRAF6 and wild type (Left) or PxExxDAxAxxA mutant (Right) GAL4-NCoR516-811 plasmids. Immunoprecipitation was performed either with anti-Myc, anti-GAL4, or control IgG, and blotted with anti-GAL4, anti-TRAF6, or anti-Myc Ab. (E) ChIP experiment showing rapid and transient recruitment of TRAF6 and Ubc13 to the BMP7 promoter in response to IL-1β in MCF7 pretreated with E2 for 1 h. (F) Validation of siRNA efficiency in MCF7 cells. Cells are transfected with siRNA (siCtl, siTRAF6, or siUbc13) for 48 h and relative expression measured by RTqPCR, with normalization to b-actin. (G) BMP7 derepression by IL-1β, as measured by RT-PCR, requires both TRAF6 and Ubc13. MCF7 cells transfected with either control, TRAF6, or Ubc13 siRNA, were treated with E2 and/or IL-1β for 6 h. (H) Overexpression of dominant-negative TRAF6 or mutated TAB2 abrogates derepression by IL-1β in U2OS-ERα cells transiently transfected with BMP7promoter reporter and treated with E2 and vehicle (PBS) or IL-1β for 36 h. Luciferase activity was assayed and normalized to b-gal activity. (I) MEKK1’s ubiquitin-interacting motif (UIM) interacts with K63-linked, but not K48-linked, polyubiquitin chains. GST and GST-MEKK1-UIM proteins were expressed in bacteria, purified, and used for GST pulldown with recombinant K63- (Left) or K48-linked (Right) polyubiquitin chains, followed by western blotting with anti-ubiquitin or anti-GST Ab. (J) Ubc13 is required for accumulation of K63-linked polyubiquitin chains and for the recruitment of MEKK1 to the BMP7 promoter in MCF7 cells transfected with control or Ubc13 siRNA and pretreated with E2 for 1 h prior to treatment with IL-1β. ChIP was performed with anti-K63-ubiquitin or anti-MEKK1 Ab or protein A beads alone as control. Data are represented as mean ± SEM. *P < 0.05, **P < 0.01 vs. vehicle control, Student’s t test.

Article Snippet: Commercial antibodies used for western blotting, immunoprecipitation, and ChIP experiments include HDAC3 rabbit polyclonal antibody #ab7030 (Abcam); anti- HDAC3(H- 99) rabbit polyclonal antibody #sc- 11417 (Santa Cruz Biotechnology); anti- ubiquitin mouse monoclonal antibody (P4D1 clone, Cell Signaling Technology); anti- β- tubulin mouse monoclonal antibody #T0198 (Sigma- Aldrich); anti- HDAC2 rabbit polyclonal antibody #ab16032 (Abcam); anti- TAB2 mouse monoclonal antibody sc- 398188, goat polyclonal antibody sc- 11850, and rabbit polyclonal sc- 20756 (Santa Cruz Biotechnology); anti- K63 ubiquitin rabbit monoclonal antibody #05- 1308 (Millipore); anti- TRAF6 rabbit polyclonal antibody sc- 7221 (Santa Cruz Biotechnology); anti- Ubc13 rabbit polyclonal antibody #10243- 1- AP (ProteinTech); anti- Ubc13 (131- 148) rabbit polyclonal antibody #PA1- 41188 (Thermo Scientific); anti- FLAG mouse monoclonal antibody, clone M2, #F3165, and anti- Flag- HRP #A8592 (Sigma- Aldrich); antiMYC(9E10) mouse monoclonal antibody #sc- 40 (Santa Cruz Biotechnology); and anti- GST(56C1) mouse monoclonal antibody #sc- 80998 (Santa Cruz Biotechnology).

Techniques: Western Blot, Translocation Assay, Control, Immunoprecipitation, Sequencing, Transfection, Mutagenesis, Biomarker Discovery, Expressing, Reverse Transcription Polymerase Chain Reaction, Over Expression, Dominant Negative Mutation, Luciferase, Activity Assay, Ubiquitin Proteomics, Bacteria, Purification, Recombinant

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Nonproteolytic ubiquitination regulates chromatin occupancy by the NCoR/SMRT/HDAC3 corepressor complex in MCF-7 breast cancer cells.

doi: 10.1073/pnas.2502805122

Figure Lengend Snippet: Fig. 6. Graphical abstract representation of TRAF6 nuclear translocation and recruitment to the TRAF-interaction domain (TID) on NCoR/SMRT in response to IL-1β (red arrows). Within the NCoR/SMRT complex, GPS2 maintains TRAF6 activity under negative regulation (black solid line), while HDAC3 and TAB2 were identified as targets of TRAF6-mediated ubiquitination (green arrows). Dotted black arrows represent previously reported interactions between components of the NCoR/SMRT corepressor complex and associated TFs. RD1, RD2, and RD3 represent repressor domains; DAD is the deacetylase activation domain, while interaction with recruiting TFs occurs through the C’-terminal nuclear receptor–interacting domains I and II.

Article Snippet: Commercial antibodies used for western blotting, immunoprecipitation, and ChIP experiments include HDAC3 rabbit polyclonal antibody #ab7030 (Abcam); anti- HDAC3(H- 99) rabbit polyclonal antibody #sc- 11417 (Santa Cruz Biotechnology); anti- ubiquitin mouse monoclonal antibody (P4D1 clone, Cell Signaling Technology); anti- β- tubulin mouse monoclonal antibody #T0198 (Sigma- Aldrich); anti- HDAC2 rabbit polyclonal antibody #ab16032 (Abcam); anti- TAB2 mouse monoclonal antibody sc- 398188, goat polyclonal antibody sc- 11850, and rabbit polyclonal sc- 20756 (Santa Cruz Biotechnology); anti- K63 ubiquitin rabbit monoclonal antibody #05- 1308 (Millipore); anti- TRAF6 rabbit polyclonal antibody sc- 7221 (Santa Cruz Biotechnology); anti- Ubc13 rabbit polyclonal antibody #10243- 1- AP (ProteinTech); anti- Ubc13 (131- 148) rabbit polyclonal antibody #PA1- 41188 (Thermo Scientific); anti- FLAG mouse monoclonal antibody, clone M2, #F3165, and anti- Flag- HRP #A8592 (Sigma- Aldrich); antiMYC(9E10) mouse monoclonal antibody #sc- 40 (Santa Cruz Biotechnology); and anti- GST(56C1) mouse monoclonal antibody #sc- 80998 (Santa Cruz Biotechnology).

Techniques: Translocation Assay, Activity Assay, Ubiquitin Proteomics, Histone Deacetylase Assay, Activation Assay

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

Article Title: Renal collecting duct epithelial cells react to pyelonephritis-associated Escherichia coli by activating distinct TLR4-dependent and -independent inflammatory pathways.

doi: 10.4049/jimmunol.177.7.4773

Figure Lengend Snippet: FIGURE 7. Differential activation of TRAF2, TRAF6, and MAPKs by the UPEC AL511 strain in Lpsn and Lpsd MCD cells. Western blot analyses of the time-dependent expression of TRIF-, TRAF6-, and -actin-labeled, phosphorylated (p-) and total p38-la- beled, and ERK1/2-labeled bands (A) and TRAF2, -actin, and phosphorylated (p-) and total ASK1 and JNK (B) in cultured Lpsn and Lpsd MCDs incubated with AL511 (5 105 bacteria per well) for 3 h. C, Bars are mean ratio values (arbitrary units) of densitometric analyses of phosphorylated (p-) over total p38-, ERK1/ 2-, ASK1-, and JNK-labeled bands and TRAF2 or TRAF6 over -actin-labeled bands. Values are means SEM from three or four separate cultures of MCDs dissected from the kidneys of 2–4 mice in each group tested. , p 0.05 vs time 0 values.

Article Snippet: Abs against IkB- , p38, TRAF2, MAPK-activated protein kinase 2 (MAPKAPK-2), c-Jun (Cell Signaling Technology), TRIF (Imgenex), JNK, TRAF6, apoptosis signal regulatory kinase 1 (ASK1), ERK1/2 (Santa Cruz Biotechnology), and -actin (Sigma-Aldrich) were used to detect the corresponding Ags.

Techniques: Activation Assay, Western Blot, Expressing, Labeling, Cell Culture, Incubation, Bacteria