Journal: PLoS Genetics

Article Title: Rad52 Sumoylation Prevents the Toxicity of Unproductive Rad51 Filaments Independently of the Anti-Recombinase Srs2

doi: 10.1371/journal.pgen.1003833

Figure Lengend Snippet: (A) Serial 10-fold dilutions of haploid strains with the indicated genotypes were plated onto rich media (YPD) containing different MMS concentrations. rad52-L264P * denotes the original isolated mutant strain and rad52-L264P ** denotes a strain in which the RAD52 gene was replaced by the mutant newly generated by directed mutagenesis. (B) Conservation of a motif comprising L264. The primary structure of Rad52 is schematized showing the conserved N-terminus moiety containing the major DNA binding and self-association domains (black, amino acids 1 to 179) as well as the C-terminus part (white, amino acids 180 to 471) containing the RPA (amino acids 275 to 278) and the Rad51 (amino acids 376 to 379) binding domains. The alignment of the Rad52 protein in Hemiascomycetes species shows the conservation of a domain containing the non-essential L264 residue and the QDDD residues essential for RPA binding and consequently for Rad52 mediator activity. The color code used in the alignment follows the default ClustalX color scheme as implemented in JalView (see Material and Methods). Cyan is for fully hydrophobic (I, L, V, M, F), turquoise for aromatic residues containing polar moieties (Y, H), green for small polar (T, S), purple for acidic (D, E), orange for glycine (G) and dark yellow for proline (P) residues.

Article Snippet: The whole cell extract (1 mg) was incubated for 1 hour at 4°C, either with a rabbit anti-Rad52 polyclonal antibody (a gift from S. Jentsch's lab), or with 1 μg of a rabbit anti-Rad51 polyclonal antibody (Santa Cruz Biotechnology).

Techniques: Isolation, Mutagenesis, Generated, Binding Assay, Residue, Activity Assay

Journal: PLoS Genetics

Article Title: Rad52 Sumoylation Prevents the Toxicity of Unproductive Rad51 Filaments Independently of the Anti-Recombinase Srs2

doi: 10.1371/journal.pgen.1003833

Figure Lengend Snippet: (A to E) Spot assay of haploid cells with the indicated genotype on rich medium (YPD) containing increasing MMS concentrations. Note that the deletion of the SIZ2 gene (A) and the rad52-3KR allele (B) cannot suppress the MMS sensitivity of srs2 Δ cells. Therefore, the MMS resistance of rad52-L264P siz2 Δ srs2 Δ and rad52-3KR-L264P srs2 Δ cells is only related to rad52-L264P . (D) Note that the haploid strain spotted at the bottom contains both rad52-3KR and RAD52 alleles.

Article Snippet: The whole cell extract (1 mg) was incubated for 1 hour at 4°C, either with a rabbit anti-Rad52 polyclonal antibody (a gift from S. Jentsch's lab), or with 1 μg of a rabbit anti-Rad51 polyclonal antibody (Santa Cruz Biotechnology).

Techniques: Spot Test

Journal: PLoS Genetics

Article Title: Rad52 Sumoylation Prevents the Toxicity of Unproductive Rad51 Filaments Independently of the Anti-Recombinase Srs2

doi: 10.1371/journal.pgen.1003833

Figure Lengend Snippet: (A) Tetrad analysis of crosses between haploid rad52-L264P srs2 Δ strains and haploid mutants synthetically lethal with srs2 Δ. Double mutant spores, which do not contain rad52-L264P , are indicated by white squares. The white circles mark triple mutants. (B) siz2 Δ and rad52-3KR do not suppress the synthetic lethality of srs2 Δ sgs1 Δ and srs2 Δ rrm3 Δ mutants. In crosses involving siz2 Δ, white squares display spores of srs2 Δ sgs1 Δ or srs2 Δ rrm3 Δ genotypes and white circles indicate triple mutants. To analyze the genetic interaction between rad52-3KR inserted at the URA3 locus and the synthetically lethal rrm3 Δ srs2 Δ double mutant, diploids homozygous for rad52 Δ were sporulated, in order to avoid the co-expression of RAD52 and rad52-3KR . The white square indicates srs2 Δ rrm3 Δ rad52 Δ triple mutants and white circles indicate unviable srs2 Δ rrm3 Δ rad52 Δ ura3 :: rad52-3KR monosporic colonies.

Article Snippet: The whole cell extract (1 mg) was incubated for 1 hour at 4°C, either with a rabbit anti-Rad52 polyclonal antibody (a gift from S. Jentsch's lab), or with 1 μg of a rabbit anti-Rad51 polyclonal antibody (Santa Cruz Biotechnology).

Techniques: Mutagenesis, Expressing

Journal: PLoS Genetics

Article Title: Rad52 Sumoylation Prevents the Toxicity of Unproductive Rad51 Filaments Independently of the Anti-Recombinase Srs2

doi: 10.1371/journal.pgen.1003833

Figure Lengend Snippet: The HO endonuclease was induced in WT or srs2 Δ cells that express Rad52-FLAG, Rad52-L264P-FLAG or Rad52-SUMO-FLAG to create a single DSB that can be repaired by SSA. Samples were taken before induction and at 2, 4, 6 and 8 hours after galactose addition. Antibodies against the RPA complex, the FLAG epitope or Rad51 were used to precipitate protein-bound chromatin. Quantitative PCR was performed with primers located at 0.6 kb or 7.6 kb from the DSB site, using the immunoprecipitated chromatin (IP) and input DNA as template. As a control, primers specific for the ARG5,6 locus were used. The relative enrichment represents the ratio of the PCR enrichment in the IP fraction to the input fraction. The median value of at least 3 experiments is shown and error bars represent the upper and lower values observed.

Article Snippet: The whole cell extract (1 mg) was incubated for 1 hour at 4°C, either with a rabbit anti-Rad52 polyclonal antibody (a gift from S. Jentsch's lab), or with 1 μg of a rabbit anti-Rad51 polyclonal antibody (Santa Cruz Biotechnology).

Techniques: FLAG-tag, Real-time Polymerase Chain Reaction, Immunoprecipitation, Chromatin Immunoprecipitation, Control

Journal: PLoS Genetics

Article Title: Rad52 Sumoylation Prevents the Toxicity of Unproductive Rad51 Filaments Independently of the Anti-Recombinase Srs2

doi: 10.1371/journal.pgen.1003833

Figure Lengend Snippet: (A) To test the interaction with RPA, Rad52 or Rad52-L264P was immunoprecipitated with a rabbit anti-Rad52 polyclonal antibody from 1 mg of whole cell extracts (without DNAse treatment) prepared from RAD52 , rad52-L264P or rad52 Δ strains. To test the robustness of the interaction, increasing NaCl concentrations were added to the cell extracts. Proteins from the whole extracts (50 µg) and from the immunoprecipitated fractions were separated by SDS-PAGE and immunoblotted with rabbit anti-Rad52 polyclonal antibody or rabbit anti-RPA polyclonal antibody (allowing detection of the Rfa1 subunit of RPA). The signals corresponding to immunoprecipitated Rad52 or Rad52-L264P were quantified in three independent experiments and plotted as a fraction of the signal intensity measured in the 150 mM NaCl experiment. (B) To assess the interaction between Rad52-L264P and Rad51, Rad51 was immunoprecipitated from 1 mg of whole cell extracts (without DNAse treatment) from cells expressing Rad52-FLAG or Rad52-L264P-FLAG. The strength of the interaction was also evaluated against increasing NaCl concentrations. The proteins from whole cell extracts (50 µg) and from immunoprecipitated fractions were separated by SDS-PAGE and immunoblotted with rabbit anti-Rad51 polyclonal antibody and mouse anti-FLAG monoclonal antibody. The presence of Rad51 in the immunoprecipitated fraction cannot be detected to validate the efficiency of the immunoprecipitation because it migrates at the same level as the IgG anti-Rad51 used for the immunoprecipitation. However, the absence of Rad52 in the rad51 Δ immunoprecipitate confirmed that the Rad52-FLAG signals observed are related to the Rad52-Rad51 interaction. The signals corresponding to immunoprecipitated Rad52 or Rad52-L264P were quantified in three independent experiments and plotted as in (A).

Article Snippet: The whole cell extract (1 mg) was incubated for 1 hour at 4°C, either with a rabbit anti-Rad52 polyclonal antibody (a gift from S. Jentsch's lab), or with 1 μg of a rabbit anti-Rad51 polyclonal antibody (Santa Cruz Biotechnology).

Techniques: Immunoprecipitation, SDS Page, Expressing

Journal: PLoS Genetics

Article Title: Rad52 Sumoylation Prevents the Toxicity of Unproductive Rad51 Filaments Independently of the Anti-Recombinase Srs2

doi: 10.1371/journal.pgen.1003833

Figure Lengend Snippet: ( A ) The purity of recombinant Rad52 and Rad52-L264P (2 µg/each) was assessed by separation on 8% SDS-PAGE and staining with Coomassie blue. (B) Binding of Rad52-L264P to ssDNA. Protein titration reactions were performed by incubating 0.27 µM of a 62-nucleotides long Cy5-labeled ssDNA fragment with various amounts of Rad52-L264P at 37°C for 10 min (protein/bases: 1/10, 1/5, 1/2.5, 1/1.25, 1/0.6, 1/0.3, 1/0.15). Quantification of free ssDNA is shown. The data were fitted into a sigmoidal curve by using the PRISM software (GraphPad). (C) Rad52-L264P-mediated DNA annealing. Representative gels of Rad52 or Rad52-L264P-promoted DNA annealing are shown in the upper panel (Rad52/bases: 1/100, 1/42, 1/14, 1/6; same DNA as in (B) with reverse-complement, 340 nM each). The dsDNA/total DNA ratio at 10 min is shown in the lower panel. (D) RPA bound to ssDNA inhibits equally Rad52- and Rad52-L264P-catalyzed annealing reactions. Reactions were carried out with primers 25 and 26 (200 nM each, see Material and Methods) that were first incubated with 30 nM RPA (1/13 bases) at 30°C for 5 minutes, followed by addition of 40 nM Rad52 (1/10 bases). Self-annealing of the primers incubated without proteins and reactions performed without RPA or Rad52 are also shown. (E) Over-stimulation of DNA strand exchange by Rad52-L264P. Upper panel , diagram of the reaction substrates and products. Middle panel , ethidium bromide-stained DNA gel. As shown by the standard reaction (st), Rad51 efficiently catalyzes the formation of nicked circular products. Pre-bound RPA inhibits this reaction (line 2). Increasing amounts of Rad52 (lines 3–6, Rad52/bases: 1/55, 1/27, 1/18, 1/14) or Rad52-L264P (lines 7–10, Rad52/bases: 1/880, 1/220, 1/110, 1/55) overcome the inhibitory effect of pre-bound RPA. In line 1, only RPA was added to the reaction. Lower panel , the ratio of the nicked circular product over the sum of the linear dsDNA substrate and the nicked circular product is shown. (F) Salt titration of Rad51-Rad52/Rad52-L264P-ssDNA complex formation. The nucleoprotein complexes were assembled by incubating 0.8 µM Rad51 with 0.09 µM Rad52 or Rad52-L264P and 0.08 µM RPA pre-bound to 2.5 µM Cy5-labeled ssDNA (400 nucleotides) in the presence of the indicated NaCl concentrations at 37°C for 15 min. The Cy5 signals after nucleoprotein gel eletrophoresis are shown. Quantifications are shown below. Data were fitted into a third order polynomial curve. Western blot analysis of the gel using antibodies against Rad51 or Rad52 is also shown. Stars indicate signals corresponding to proteins not bound to ssDNA. (G) Transmission electron microscopy images of protein-DNA complexes showing the association of Rad52 with complete Rad51 filaments. Positive (left) and negative (right) staining images are shown for each type of filaments. The proportion of each type of complete Rad51 filaments formed by Rad52 or Rad52-L264P at different NaCl concentrations is shown and compared to a control reaction without Rad52. 100 molecules were examined in each experiment.

Article Snippet: The whole cell extract (1 mg) was incubated for 1 hour at 4°C, either with a rabbit anti-Rad52 polyclonal antibody (a gift from S. Jentsch's lab), or with 1 μg of a rabbit anti-Rad51 polyclonal antibody (Santa Cruz Biotechnology).

Techniques: Recombinant, SDS Page, Staining, Binding Assay, Titration, Labeling, Software, Incubation, Western Blot, Transmission Assay, Electron Microscopy, Control

Journal: PLoS Genetics

Article Title: Rad52 Sumoylation Prevents the Toxicity of Unproductive Rad51 Filaments Independently of the Anti-Recombinase Srs2

doi: 10.1371/journal.pgen.1003833

Figure Lengend Snippet: (A) Spot assay of haploid cells over-expressing SIZ2 . Serial 10-fold dilutions were plated on minimal media lacking uracil with or without MMS. Strains of the indicated genotype were transformed with an empty vector or with the same plasmid containing the SIZ2 gene. (B) Over-expression of SIZ2 stimulates Rad52 sumoylation. Proteins conjugated with a His 7 -SUMO radical were pull-down on Ni-NTA from 5 mg of extracts of RAD52 - FLAG cells over-expressing His7- SMT3 . Pull-downs were carried out from strains transformed with a SIZ2 -containing multi-copy vector or with an empty vector. Cells treated with 0.3% of MMS were also tested as a positive control of sumoylation. rad52-L264P-FLAG strains were also subjected to pull-down analysis. Proteins from the whole extracts (3 µg) and from the pull-down fractions were separated by SDS-PAGE and immunoblotted with an anti-FLAG mouse monoclonal antibody.

Article Snippet: The whole cell extract (1 mg) was incubated for 1 hour at 4°C, either with a rabbit anti-Rad52 polyclonal antibody (a gift from S. Jentsch's lab), or with 1 μg of a rabbit anti-Rad51 polyclonal antibody (Santa Cruz Biotechnology).

Techniques: Spot Test, Expressing, Transformation Assay, Plasmid Preparation, Over Expression, Positive Control, SDS Page

Journal: PLoS Genetics

Article Title: Rad52 Sumoylation Prevents the Toxicity of Unproductive Rad51 Filaments Independently of the Anti-Recombinase Srs2

doi: 10.1371/journal.pgen.1003833

Figure Lengend Snippet: (A) Schematic representation of the two kinds of toxic recombination intermediates eliminated by Srs2 in WT cells. We found that srs2 Δ haploid cells sensitivity to DNA damage is related to recombination-deficient Rad51 nucleoprotein filaments. The toxicity of such filaments disappears in rad52-L264P srs2Δ cells, or alternatively, the intermediates themselves are not formed. However, srs2 Δ cells sensitivity to DNA damage related with toxic intertwined HR intermediates cannot be suppressed by this allele. Unproductive Rad51 filaments can be formed after resection of a DSB located in a unique sequence in the genome. In a situation where homologous dsDNA cannot be found by the recombinase, Srs2 is essential to remove Rad51 filaments to allow alternative repair pathways such as SSA. Srs2 could also edit Rad51 filaments improperly nucleated by Rad52. In srs2 Δ cells, nonrecombinogenic Rad51 filaments could also accumulate on ssDNAs generated from the uncoupling between the helicase complex opening replicative dsDNA and the DNA synthesis machinery in replicative mutants such as mrc1 Δ. Finally, when a stable paranemic joint cannot be processed from a plectonemic joint because of mutations in genes involved in late recombination steps, Srs2 is necessary to address lesions to other DNA repair pathways. Intertwined recombination intermediates that occur between homologous chromosomes in diploid cells or between ectopic chromosomes in haploid cells cannot be suppressed by rad52-L264P . (B) Unproductive Rad51 filaments mediated by Rad52-SUMO are not toxic even in srs2 Δ cells. When mediated by Rad52, Rad51 filaments that cannot complete strand invasion have to be removed by Srs2 in order to allow SSA or post-replication repair processes (PRR). Conversely, Rad52-SUMO (or Rad52-L264P) might lower (or shorten) Rad51 filaments. These modified mediators might also change Rad51 filament properties as indicated by Rad52 occupancy on Rad51 filaments. These changes might suppress Rad51 filaments toxicity, thereby bypassing the need for Srs2. Rad51 filaments might be removed by a Srs2-independent process.

Article Snippet: The whole cell extract (1 mg) was incubated for 1 hour at 4°C, either with a rabbit anti-Rad52 polyclonal antibody (a gift from S. Jentsch's lab), or with 1 μg of a rabbit anti-Rad51 polyclonal antibody (Santa Cruz Biotechnology).

Techniques: Sequencing, Generated, DNA Synthesis, Modification

Journal: iScience

Article Title: IFRD1 is required for maintenance of bladder epithelial homeostasis

doi: 10.1016/j.isci.2024.111282

Figure Lengend Snippet: Ifrd1 −/− mice display increased urothelial cell death, dysregulated renewal and aberrant voiding behavior (A) TUNEL staining (green) of WT and Ifrd1 −/− urothelial cells. Nuclei are stained with DAPI (blue) ( n = 3/group). Scale bar, 100 μm. (B) DHE staining (red) of WT and Ifrd1 −/− urothelium. Nuclei are stained with DAPI (blue). Quantitation of DHE staining intensity ( n = 3 bladders/group; data shown as mean (±SD); p values by unpaired t test). Scale bar, 100 μm. (C) Representative TEM (left) and urine cytology (right) images show exfoliated bladder epithelial cells. Quantitation of exfoliated/shed epithelial cells ( n = 5 WT, 4 Ifrd1 −/− mice; data shown as mean (±SD); p values by unpaired t test). Scale bar, 2 μm. (D) Immunostaining for Ki-67 (red) and E-cadherin (green) in WT and Ifrd1 −/− urothelial cells ( n = 3/group). Scale bar, 100 μm. (E) Immunostaining of CDKN1B (red) in WT and Ifrd1 −/− urothelium ( n = 3/group). Scale bar, 50 μm. (F) RT-qPCR analysis of Cdkn1b transcripts in WT and Ifrd1 −/− bladders ( n = 6 WT, 3 Ifrd1 −/− bladders; data shown as mean (±SD); p values by Mann-Whitney test). (G) FITC-dextran permeability assay (green) in the WT and Ifrd1 −/− urothelial cells reveal superficial cells with intracellular FITC-dextran in the Ifrd1 −/− urothelium. Nuclei are stained with DAPI (blue) ( n = 3/group). Quantitation of FITC-dextran staining intensity ( n = 3 bladders/group; data shown as mean (±SD); p values by Mann-Whitney test). Scale bar, 100 μm. (H) Representative images of WT (left) and Ifrd1 −/− (right) void spot assay (VSA). (I) Quantitation of the area of total voids from the VSA. Each dark dot is the average area of all the individual voids that are depicted as transparent dots of the same color. ( n = 4/group, data shown as mean (±SEM), p values by unpaired t test). (J) Quantitation of the size of all the individual spots from the VSA. All the data points from one mouse are presented in one color. (Data represented as interquartile range of size distribution where the central line represents the median (±SD), n = 4/group, p values by unpaired t test).

Article Snippet: 50 μl of 10 mg/ml of fluorescein isothiocyanate (FITC)-conjugated-dextran (FITC-dextran, 10000MW, Cat. No. D1820, Invitrogen) in 1X PBS was injected transurethrally into the bladder lumen.

Techniques: TUNEL Assay, Staining, Quantitation Assay, Immunostaining, Quantitative RT-PCR, MANN-WHITNEY, FITC-Dextran Permeability Assay, Spot Test

Journal: iScience

Article Title: IFRD1 is required for maintenance of bladder epithelial homeostasis

doi: 10.1016/j.isci.2024.111282

Figure Lengend Snippet:

Article Snippet: 50 μl of 10 mg/ml of fluorescein isothiocyanate (FITC)-conjugated-dextran (FITC-dextran, 10000MW, Cat. No. D1820, Invitrogen) in 1X PBS was injected transurethrally into the bladder lumen.

Techniques: Recombinant, Lysis, Electron Microscopy, Staining, In Situ, TUNEL Assay, Gene Expression, Generated, Software, Microscopy

Journal: STAR Protocols

Article Title: GST-Perfringolysin O production for the localization and quantification of membrane cholesterol in human and mouse brain and liver

doi: 10.1016/j.xpro.2021.101068

Figure Lengend Snippet:

Article Snippet: Mouse IgG 1 GST (A-6) (Immunofluorescence: 1:200 for human samples; 1:250 for mouse samples. Protein-lipid assay: 1:1000) , Santa Cruz , Cat#374171.

Techniques: Immunofluorescence, Virus, Recombinant, Protease Inhibitor, Generated, Lysis, Saline, Plasmid Preparation, Sequencing, Software, Confocal Microscopy, Construct, Spectrophotometry, Microscopy

Journal: Nature Communications

Article Title: Zinc is a potent and specific inhibitor of IFN-λ3 signalling

doi: 10.1038/ncomms15245

Figure Lengend Snippet: ( a ) Huh-7 cells were treated with 50 U ml −1 IFN-α or 100 ng ml −1 IFN-λ3 in combination with increasing amounts of zinc (0, 5 and 50 μM ZnSO 4 ) or 5 μM ZnSO 4 in combination with 10 mg ml −1 or 50 mg ml −1 BSA to sequester zinc. STAT1 phosphorylation 30 min post IFN-λ3 treatment was dose-dependently inhibited by ZnSO 4 , and reversed with albumin. IFN-α treatment however, was unaffected. ( b ) The expression of IFN-λ3-induced ISGs, MX1 and viperin , were affected in a similar manner, with minimal effects on IFN-α-mediated ISG expression (Welch's t -test). ( c ) To determine if zinc inhibits the antiviral response, Huh-7 cells infected with the JFH-1 strain of HCV and H1N1 influenza virus were treated with IFN-α and IFN-λ3 in combination with zinc. IFN-α-reduced JFH-1 intracellular RNA by ∼75% independent of zinc concentration, however the antiviral activity of IFN-λ3 was significantly reduced from ∼50% to ∼20% with 50 μM ZnSO 4 . The antiviral activity of IFN-α against influenza virus was only partially inhibited by zinc, however a similar dose-dependent inhibition of IFN-λ3 antiviral activity was observed, almost ablating its effects at 50 μM ZnSO 4 (Welch's t -test). Data are representative of two (western blotting) and three (gene expression) independent experiments. * P <0.05, ** P <0.01, *** P <0.001, (mean±s.e.). UT, untreated.

Article Snippet: Protein was transferred on to nitrocellulose membrane, and β-actin and STAT1 were probed using STAT1 (Santa Cruz Biotechnology, SC-345 clone C-24, rabbit anti-human, 1:2,000 dilution), Tyr701 p-STAT1 (Cell Signalling, 9167 clone 58D6, rabbit anti-human, 1:500 dilution) and β-actin (Sigma-Aldrich, A1978 clone AC-15, mouse anti-human, 1:10,000 dilution) antibodies.

Techniques: Phospho-proteomics, Expressing, Infection, Virus, Concentration Assay, Activity Assay, Inhibition, Western Blot, Gene Expression

Journal: Nature Communications

Article Title: Zinc is a potent and specific inhibitor of IFN-λ3 signalling

doi: 10.1038/ncomms15245

Figure Lengend Snippet: Zinc-regulated transcriptome disease class and biological process as identified by DAVID functional annotation.

Article Snippet: Protein was transferred on to nitrocellulose membrane, and β-actin and STAT1 were probed using STAT1 (Santa Cruz Biotechnology, SC-345 clone C-24, rabbit anti-human, 1:2,000 dilution), Tyr701 p-STAT1 (Cell Signalling, 9167 clone 58D6, rabbit anti-human, 1:500 dilution) and β-actin (Sigma-Aldrich, A1978 clone AC-15, mouse anti-human, 1:10,000 dilution) antibodies.

Techniques: Functional Assay, Infection, Transduction

Journal: Nature Communications

Article Title: Zinc is a potent and specific inhibitor of IFN-λ3 signalling

doi: 10.1038/ncomms15245

Figure Lengend Snippet: ( a ) To ascertain the cellular location of zinc inhibition following ZnSO 4 treatment, Huh-7 cells were pre-treated for 24 h with 50 μM ZnSO 4 , washed thoroughly, and then treated with IFN-λ3 ±50 μM ZnSO 4 for 15 min. STAT1 phosphorylation was significantly inhibited only when IFN-λ3 was administered in the presence of zinc, suggesting that zinc inhibits IFN-λ3 signalling at the receptor. ( b ) To identify cytokine zinc binding, 200 ng of IFN-α or IFN-λ3 were added to a zinc loaded IMAC-affinity resin for 2 h, after which bound protein was examined by SDS–PAGE. Dimeric forms of IFN-α and IFN-λ3 bound the zinc resin, as did monomeric IFN-λ3. ( c ) Using protein G beads, Co-IP was performed on Huh-7 cell lysates using recombinant IFN-λ3 alone or with the addition of 50 μM ZnSO 4 . Co-IP of both IFN-λ3 (66% reduction) and IFNLR1 (40% reduction) was reduced in the presence of zinc, when IFNLR1 and IFN-λ3 were immunoprecipitated respectively, demonstrating a direct inhibition of receptor:cytokine interaction by zinc. ( d ) Co-IP studies were confirmed using proximity ligation assays to examine the interaction of IFN-λ3 and IFNLR1 in the presence of zinc in situ . Confocal microscopy of IFN-λ3 treated Huh-7 cells demonstrated a reduction in fluorescent signals (red fluorescent spots), indicative of IFNLR1:IFN-λ3 interaction (within 40 nm). No signal was observed within the negative control assay lacking the primary IFN-λ3 antibody (1° Ab) or lacking the addition of IFN-λ3. ( e ) Proximity ligation signals per cell were quantified using ImageJ software, demonstrating a 50% decrease in IFNLR1:IFN-λ3 interactions per cell (Mann–Whitney test). ( d ) Scale bars, 10 μm. Data are representative of two independent experiments, *** P <0.001, (mean±s.e.).

Article Snippet: Protein was transferred on to nitrocellulose membrane, and β-actin and STAT1 were probed using STAT1 (Santa Cruz Biotechnology, SC-345 clone C-24, rabbit anti-human, 1:2,000 dilution), Tyr701 p-STAT1 (Cell Signalling, 9167 clone 58D6, rabbit anti-human, 1:500 dilution) and β-actin (Sigma-Aldrich, A1978 clone AC-15, mouse anti-human, 1:10,000 dilution) antibodies.

Techniques: Inhibition, Phospho-proteomics, Binding Assay, SDS Page, Co-Immunoprecipitation Assay, Recombinant, Immunoprecipitation, Ligation, In Situ, Confocal Microscopy, Negative Control, Software, MANN-WHITNEY

Journal: Frontiers in Pharmacology

Article Title: Ginger Constituent 6-Shogaol Inhibits Inflammation- and Angiogenesis-Related Cell Functions in Primary Human Endothelial Cells

doi: 10.3389/fphar.2022.844767

Figure Lengend Snippet: LPS-induced NFκB promotor activity is blocked by 6-shogaol but remains unimpaired by 6-gingerol. (A,B) 1 × 10 6 HUVECs per aluminum cuvette were transiently transfected with a Renilla luciferase control plasmid pGL4.74[hRluc/TK] and firefly luciferase reporter plasmid pGL4.32[luc2P/NF-κB-RE/Hygro] using a Nucleofector 2b device. 24 h after transfection, the cells were treated with indicated concentrations of (A) 6-shogaol or (B) 6-gingerol for 6 h before NFκB promotor activity was induced by LPS (1 μg/ml) for an additional 4 h. NFκB promotor activity was measured using a luminometer. (C) Confluent HUVECs were treated with 30 µM of 6-shogaol before the cells were activated with LPS (1 μg/ml) for the indicated time points. Subsequently, the cells were stained for p65, and fluorescence microscopy images were analyzed using ImageJ software. For each condition, one representative image out of four independently performed experiments is shown. Scale bar represents 50 µm. (D) Confluent HUVECs were treated with 30 µM of 6-shogaol for indicated time points. Protein levels of TLR4 were determined by Western blot analysis. Actin served as the loading control. One representative blot of four independently performed experiments is shown. (E) Confluent HUVECs were treated with indicated increasing concentrations of 6-shogaol and were induced with LPS (1 μg/ml) for 24 h before the samples were subjected to Western blot analysis for HO-1 total protein. Actin served as the loading control. One representative Western blot is shown. 6-SG: 6-shogaol. Data are expressed as mean ± SD; (A,B) n = 3; * p ≤ 0.05 vs. LPS; (C,D) n = 4; (E) n = 5; * p ≤ 0.05 vs. LPS.

Article Snippet: After blocking unspecific binding sites using 0.2% BSA (MilliporeSigma, Burlington, MA, United States), the cells were incubated in a 0.2% BSA solution containing the primary antibody anti-NFκB-p65 (rabbit, anti-human, 1:400, sc-372; Santa Cruz Biotechnology, Dallas, TX, United States).

Techniques: Activity Assay, Transfection, Luciferase, Control, Plasmid Preparation, Staining, Fluorescence, Microscopy, Software, Western Blot