Journal: Molecular immunology

Article Title: Desialylation of airway epithelial cells during influenza virus infection enhances pneumococcal adhesion via galectin binding

doi: 10.1016/j.molimm.2014.12.010

Figure Lengend Snippet: Galectin-mediated adhesion of S. pneumoniae to airway epithelial cells and binding to capsular polysaccharides A549 cells were grown in ELISA plates and treated with bacterial neuraminidase ( Arthrobacter ureafaciens and Clostridium perfringens; Neu K). The cells were exposed for 1h to Sp3 (MOI 10) alone or Sp3 previously incubated with 15 µg/ml of rhGal1 or rhGal3. Bacterial binding to the cells was assessed using a primary anti- Streptococcus pneumoniae antibody, followed by an HRP-conjugated secondary antibody. The bacterial adhesion to cells in the presence of galectins was normalized to the bacterial adhesion in the absence of exogenous galectin. Data shown and the bar graphs are representative data from at least three independent experiments. *p

Article Snippet: After washed, the membranes were incubated with anti-galectin antibodies followed by an HRP-conjugated secondary antibody (Thermo Scientific) and the results were visualized with ECL reagents.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Incubation

Journal: Molecular immunology

Article Title: Desialylation of airway epithelial cells during influenza virus infection enhances pneumococcal adhesion via galectin binding

doi: 10.1016/j.molimm.2014.12.010

Figure Lengend Snippet: In vitro effect of PR8 and neuraminidase exposure of A549 cells on expression and binding of galectins to A549 cells Exposure of A549 cells to PR8 (A) or neuraminidase (B) modulates galectin expression (A1 and B1) and secretion (A2 and B2). A549 cells were infected with PR8 (MOI 5) for 72h (A), or treated with a bacterial neuraminidase cocktail ( Arthrobacter ureafaciens and Clostridium perfringens; Neu K) (B). Galectin (Gal1 and Gal3) expression or secretion was assessed from cell lysates (A1 and B1) or culture supernatant (A2 and B2) with Western blot. Bar graphs show fold changes of galectin expression from PR8-infected or neuraminidase-treated cells (Neu K) in comparison with untreated cells (ctrl) normalized to tubulin. (C) Neuraminidase-treated (Neu K) or untreated (ctrl) cells were incubated with 15 µg/ml of biotinylated rhGal1, rhGal3, or galectins in the presence of lactose (0.1M) (Gal1+lac, Gal3+lac), followed by streptavidin-APC for flow cytometry analysis. The exogenous galectin binding to A549 cells was normalized to A549 cells without exogenous galectin (ctrl). (D) Effects of influenza viral neuraminidase (Neu N2) and bacterial neuraminidases ( Arthrobacter ureafaciens and Clostridium perfringens; Neu K) on galectin binding to A549 cells. A549 cells grown in ELISA plates were subject to neuraminidase treatment (Neu K or Neu N2), followed by incubation with 15 µg/ml of exogenous rhGal1 or rhGal3. Galectin binding to the cells was assessed using primary antibodies against Gal1 or Gal3 followed by an HRP-conjugated secondary antibody. The galectin binding levels to neuraminidase-treated cells were normalized to the binding to the untreated cells (ctrl). Data shown and the bar graphs are representative data from at least three independent experiments. *p

Article Snippet: After washed, the membranes were incubated with anti-galectin antibodies followed by an HRP-conjugated secondary antibody (Thermo Scientific) and the results were visualized with ECL reagents.

Techniques: In Vitro, Expressing, Binding Assay, Infection, Western Blot, Incubation, Flow Cytometry, Cytometry, Enzyme-linked Immunosorbent Assay

Journal: Molecular immunology

Article Title: Desialylation of airway epithelial cells during influenza virus infection enhances pneumococcal adhesion via galectin binding

doi: 10.1016/j.molimm.2014.12.010

Figure Lengend Snippet: Binding of galectins to influenza PR8 virion components and effect of neuraminidase exposure (A) Binding of rhGal1 and rhGal3 to influenza hemagglutinin (HA0, precursor HA containing a hydrophobic signal sequence; HA1 and HA2, subunits of HA). Lysates from PR8 virus were prepared and subjected to Western blot. The total viral proteins were revealed by an α-PR8 antibody, and the viral HA by an α-HA antibody. Galectin binding was performed by overlaying membrane with rhGal1, rhGal3, and the specificity of the binding assessed by preincubation of the galectins with lactose (0.2 M) (rhGal1+lac, rhGal3+lac), followed by anti-Gal1 or anti-Gal3 antibodies, followed by HRP-conjugated secondary antibodies. To confirm the carbohydrate specificity of the galectin-HA interactions, HA was treated with PNGase F (HA* in a) or β-galactosidase (HA** in b or c) and subjected to lectin blot (a and c) as described above, or ELISA (b). (B) SPR sensogram of binding rhGal1 to HA. SPR was measured with immobilized rhGal1 and using HA (left panel) or desialylated HA (right panel) as analytes. HA starts at 12.5µg/ml, with 2-fold serial dilution; asialo-HA starts at 6.25 µg/ml. (C) SPR sensogram of binding of rhGal3 to HA. SPR was measured with immobilized rhGal3 and using HA (left panel) or desialylated HA (right panel) as analytes. HA starts at 50 µg/ml, with 2-fold serial dilution; asialo-HA starts at 6.25 µg/ml.

Article Snippet: After washed, the membranes were incubated with anti-galectin antibodies followed by an HRP-conjugated secondary antibody (Thermo Scientific) and the results were visualized with ECL reagents.

Techniques: Binding Assay, Sequencing, Western Blot, Enzyme-linked Immunosorbent Assay, SPR Assay, Serial Dilution

Journal: BMC Cell Biology

Article Title: Differential growth factor regulation of aspartyl-(asparaginyl)-?-hydroxylase family genes in SH-Sy5y human neuroblastoma cells

doi: 10.1186/1471-2121-7-41

Figure Lengend Snippet: Modulation of AAH, Humbug, and Junctin expression by growth factor stimulation. Subconfluent SH-Sy5y cell cultures were serum starved over night then stimulated with vehicle (CON), insulin (50 nM), IGF-1 (25 nM), or NGF (2.5 ng/ml) for 24 hours. (A) Upper panel: Representative Western blot demonstrating AAH protein expression (~86 kD) using the HBOH monoclonal antibody. Immunoreactivity was detected with horseradish peroxidase conjugated secondary antibody, ECL reagents, and digital imaging. Lower panel-blots were stripped and re-probed to detect β-actin as a loading control. The positions of molecular weight standards included in the analysis are indicated at the left. (B) AAH immunoreactivity was measured directly in 96-well micro-cultures using the microtiter immunocytochemical ELISA (MICE) assay. The MICE index corresponds to immunoreactivity corrected for cell density. (C) Directional motility was measured in response to insulin or IGF-I stimulation using the ATP Luminescence-Based Motility/Invasion (ALMI) assay. The total percentages of motile cells (motility index), both adherent and non-adherent, were calculated (see Methods). (D-F) AAH, Humbug, and Junctin mRNA levels were measured by real time quantitative RT-PCR with results normalized to 18S. See Methods section for detailed protocols. Graphs depict mean ± S.D. of results obtained from 6 or 8 replicate independent cultures. Data were analyzed using ANOVA with the Fisher Least Significant Difference post-hoc test (+P

Article Snippet: Immunoreactivity was detected with horseradish peroxidase conjugated secondary antibody (Pierce, Rockford, IL) and the TMB soluble peroxidase substrate (Pierce, Rockford, IL).

Techniques: Expressing, Western Blot, Imaging, Molecular Weight, Enzyme-linked Immunosorbent Assay, Mouse Assay, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Herpes Simplex Virus Glycoprotein C Regulates Low pH Entry

doi: 10.1101/858472

Figure Lengend Snippet: HSV-1 gC facilitates acid-induced conformational changes in the fusion protein gB. Extracellular preparations of HSV-1 gCR or ΔgC (∼ 10 7 genome copy numbers) were treated with pHs ranging from 7.3 to 5.0 and blotted directly to nitrocellulose. Blots were probed with the indicated gB MAbs H126, H1838, SS144, or H1359 at neutral pH followed by HRP-conjugated anti-mouse secondary antibody. The antibody name is shown at the left and the gB domain to which each MAb is directed is indicated in parenthesis. These are individual examples of experiments that were quantitated and averaged together with multiple similar independent determinations. Summarized quantitative results are depicted in Figure 6 .

Article Snippet: After incubation with horseradish peroxidase-conjugated secondary antibodies, enhanced chemiluminescent substrate (Thermo Fisher Scientific) was added, and blots were exposed to X-ray film (Genesee Scientific).

Techniques:

Journal: The Journal of Neuroscience

Article Title: Coassembly and Coupling of SK2 Channels and mGlu5 Receptors

doi: 10.1523/JNEUROSCI.2038-14.2014

Figure Lengend Snippet: Association of mGlu 5 receptors and SK2 channels. A , Coimmunoprecipitation of mGlu 5 receptors and SK2 channels from rat hippocampus. Solubilized extracts from rat hippocampus were subjected to immunoprecipitation analysis using rabbit anti-flag antibody (2 μg; lane 1), rabbit anti-mGlu 5 receptor antibody (2 μg; lane 2), and rabbit anti-SK2 channel antibody (2 μg; lane 3). Extracts (Crude) and immunoprecipitates (IP) were analyzed by SDS-PAGE and immunoblotted (IB) using a rabbit anti-mGlu 5 receptor antibody (1 μg/ml) or guinea pig anti-SK2 channel antibody (1 μg/ml). An HRP-conjugated anti-rabbit IgG TrueBlot (1:1000; IB: anti-mGlu 5 ) or an HRP-conjugated goat anti-guinea pig IgG (1:30,000; IB: anti-SK2) was used as a secondary antibody. The immunoreactive bands were visualized by chemiluminescence. B , Codistribution of mGlu 5 receptors and SK2 channels expressed in HEK-293T cells. Cells were transiently transfected with the cDNAs encoding SK2 CFP channels plus the mGlu 5 YFP receptors and analyzed by double fluorescence with a confocal microscope. Superimposition of images (merge) reveals a high codistribution of SK2 CFP channels and the mGlu 5 YFP receptors. Scale bar, 10 μm. C , Coimmunoprecipitation of mGlu 5 receptors and SK2 channels from HEK-293T cells. Cells were transiently transfected with SK2 YFP/CFP channels (lane1), SK2 YFP/CFP channels plus mGlu 5 receptors (lane2), or mGlu 5 receptors (lane 3) and processed for immunoprecipitation using mouse anti-GFP antibody (1 μg; IP: anti-SK2) or rabbit anti-mGlu 5 receptor antibody (1 μg; IP: anti-mGlu 5 ). Extracts (Crude) and immunoprecipitates (IP) were analyzed by SDS-PAGE and immunoblotted (IB) using a rabbit anti-SK2 channel antibody (1 μg/ml; IB: anti-SK2) or a rabbit anti-mGlu 5 receptor antibody (1 μg/ml; IB: anti-mGlu 5 ). An HRP-conjugated anti-rabbit IgG TrueBlot (1:1000) was used as a secondary antibody. The immunoreactive bands were visualized by chemiluminescence.

Article Snippet: Immune complexes were dissociated, transferred to polyvinylidene difluoride membranes and probed with the indicated primary antibodies followed by horseradish-peroxidase (HRP)-conjugated secondary antibodies (TrueBlot; 1:1000; eBioscience).

Techniques: Immunoprecipitation, SDS Page, Transfection, Fluorescence, Microscopy

Journal: Journal of Cell Science

Article Title: Caveolae provide a specialized membrane environment for respiratory syncytial virus assembly

doi: 10.1242/jcs.198853

Figure Lengend Snippet: Caveolin-1 interacts with the RSV G and M protein complex on the surface of infected cells. (A) Immunoprecipitations of RSV G, M and M2-1 proteins from HeLa whole-cell lysates of mock-infected or RSV-infected cells (22 hpi). Immunoprecipitates were probed for G protein (top panel), M protein (middle panel) and caveolin-1 (bottom panel). Caveolin-1 protein was measured by densitometry (bottom graph). (B) Streptavidin–HRP blot of lysates of surface-biotinylated HeLa cells. M, mock infected; I, RSV infected. (C) Streptavidin–HRP blot of immunoprecipitates from lysates shown in B, using antibodies against RSV G protein or caveolin-1. Representative data of two independent experiments are shown.

Article Snippet: Streptavidin conjugated to horseradish peroxidase (HRP), CellMask™Orange, phalloidin–FITC, and Alexa Fluor 488, 555, 633- and HRP-conjugated secondary antibodies were from LifeTechnology, Singapore.

Techniques: Infection

Journal: Viruses

Article Title: DNA Vaccine-Encoded Flagellin Can Be Used as an Adjuvant Scaffold to Augment HIV-1 gp41 Membrane Proximal External Region Immunogenicity

doi: 10.3390/v10030100

Figure Lengend Snippet: FliC Δ174-400 with gp41 607-683 inserted at its N-terminus is secreted and maintains TLR5 agonist activity. ( A ) Schematic representation of flagellin constructs; ( B ) Western blot of cell lysates and supernatants from transiently transfected 293T. Cells were transfected with pVAX, FliC, FliC Δ174–400, FliC Δ220–320, gp41 607–683 FliC, gp41 607–683 FliC Δ174–400, gp41 607–683 FliC Δ220–320, FliC Δ174-[gp41 607–683 ]-400, FliC Δ220-[gp41 607–683 ]-320, FliC gp41 607–683 , FliC Δ174–400 gp41 607–683 or FliC Δ220–320 gp41 607–683 . Samples were collected 48 h post transfection. Blots were probed with a mouse anti-FLAG tag antibody to detect FLAG-tagged flagellin proteins. A mouse anti-tubulin antibody, was used to detect tubulin as a loading control; ( C ) Measurement of relative secretion level of FLAG-tagged proteins using capture ELISA. Supernatants from transiently transfected 293T were applied to anti-FLAG tag antibody coated plates and probed with an HRP conjugated mouse anti-FLAG tag antibody. Results shown represent the mean of 3 different experiments where FliC is set to a value of 1. Error bars represent standard error of the mean; ( D ) Relative TLR5 agonist activity of secreted FliC proteins. Normalized supernatants from transiently transfected 293T were diluted 1:100 and added to HEK-Blue-hTLR5 cells. After 20 h incubation, the quantity of SEAP produced was determined using a colorimetric enzyme assay. Relative quantities, presented as response ratios, are indicative of TLR5 agonist activity. Results shown represent the mean of 3 different experiments where pVAX is set to a value of 1. Error bars represent standard error of the mean.

Article Snippet: Plates were subsequently washed three times with PBS-T, then incubated with a mouse anti-FLAG tag antibody conjugated to horseradish peroxidase (HRP) (Sigma, St. Louis, MO, USA) at a 1:20,000 dilution for 1 h at RT.

Techniques: Activity Assay, Construct, Western Blot, Transfection, FLAG-tag, Enzyme-linked Immunosorbent Assay, Incubation, Produced, Enzymatic Assay

Journal: Viruses

Article Title: DNA Vaccine-Encoded Flagellin Can Be Used as an Adjuvant Scaffold to Augment HIV-1 gp41 Membrane Proximal External Region Immunogenicity

doi: 10.3390/v10030100

Figure Lengend Snippet: FliC constructs expressed in a mammalian system are secreted and maintain Toll-like receptor 5 (TLR5) agonist activity. ( A ) Schematic representation of flagellin (FliC) constructs; ( B ) Western blot of cell lysates, supernatants and PNGase F treated supernatants from transiently transfected 293T. Cells were transfected with pVAX (empty vector), FliC, FliC Δ174–400 or FliC Δ220–320. Samples were collected 48 h post transfection. Blots were probed with a mouse anti-FLAG tag antibody to detect FLAG-tagged flagellin proteins. A mouse anti-tubulin antibody, was used to detect tubulin as a loading control; ( C ) Measurement of relative secretion level of FLAG-tagged proteins using capture ELISA. Supernatants from transiently transfected 293T were applied to anti-FLAG tag antibody coated plates and probed with a horseradish peroxidase (HRP) conjugated mouse anti-FLAG tag antibody. Results shown represent the mean of 4 different experiments where FliC is set to a value of 1. Error bars represent standard error of the mean; ( D ) Relative TLR5 agonist activity of secreted FliC proteins. Normalized supernatants from transiently transfected 293T were diluted 1:100 and added to HEK-Blue-hTLR5 cells. After 20 h incubation, the quantity of secreted embryonic alkaline phosphatase (SEAP) produced was determined using a colorimetric enzyme assay. Relative quantities, presented as response ratios, are indicative of TLR5 agonist activity. Results shown represent the mean of 4 different experiments where pVAX is set to a value of 1. Error bars represent standard error of the mean.

Article Snippet: Plates were subsequently washed three times with PBS-T, then incubated with a mouse anti-FLAG tag antibody conjugated to horseradish peroxidase (HRP) (Sigma, St. Louis, MO, USA) at a 1:20,000 dilution for 1 h at RT.

Techniques: Construct, Activity Assay, Western Blot, Transfection, Plasmid Preparation, FLAG-tag, Enzyme-linked Immunosorbent Assay, Incubation, Produced, Enzymatic Assay

Journal: Infection and Immunity

Article Title: Biochemical and Localization Analyses of Putative Type III Secretion Translocator Proteins CopB and CopB2 of Chlamydia trachomatis Reveal Significant Distinctions ▿

doi: 10.1128/IAI.00159-11

Figure Lengend Snippet: Temporal analysis of CopB and CopB2 levels during chlamydial infection. HeLa cells were mock infected or infected with C. trachomatis L2 at an MOI of 10 for 1 h at 37°C. Cultures were thoroughly washed, supplemented with fresh medium, and subsequently harvested at the indicated times (in hours) after the medium addition. TCA-concentrated, whole-cell culture lysates were probed via immunoblotting with rabbit anti-CopB or anti-CopB2. Antibodies specific for the T3S structural protein CdsJ and chlamydial envelope protein MOMP were used as controls. Proteins were visualized by probing with secondary antibodies conjugated to horseradish peroxidase and development with ECL Plus chemiluminescence reagent.

Article Snippet: After incubation with specific primary antibodies, immunoblots were probed with the appropriate alkaline phosphatase or horseradish peroxidase-conjugated secondary antibodies (Sigma) and developed with One Step nitroblue tetrazolium–5-bromo-4-chloro-3-indolylphosphate (NBT-BCIP; Pierce Biotechnology, Inc., Rockford, IL) or ECL Plus reagent (GE Healthcare), respectively.

Techniques: Infection, Cell Culture

Journal: Infection and Immunity

Article Title: Biochemical and Localization Analyses of Putative Type III Secretion Translocator Proteins CopB and CopB2 of Chlamydia trachomatis Reveal Significant Distinctions ▿

doi: 10.1128/IAI.00159-11

Figure Lengend Snippet: CopB and CopB2 fractionation in whole-culture membrane flotation gradients. C. trachomatis -infected (L2) or mock-infected (UI) HeLa cultures were disrupted 24 h postinfection, and membrane-containing, clarified lysates (input) were generated. Lysates were subjected to density gradient centrifugation through sucrose. Twelve individual column fractions were collected, and proteins were concentrated via TCA precipitation. Equivalent volumes of material corresponding to the top (1) to the bottom (12) fractions of the column were resolved by SDS-PAGE in 12% polyacrylamide gels. Immunoblots of input and column fractions were probed with anti-CopB and anti-CopB2 or with anti-IncG, anti-CdsD, and anti-CdsJ as fractionation controls. Proteins were visualized by probing with secondary antibodies conjugated to horseradish peroxidase and development with ECL Plus chemiluminescence reagent.

Article Snippet: After incubation with specific primary antibodies, immunoblots were probed with the appropriate alkaline phosphatase or horseradish peroxidase-conjugated secondary antibodies (Sigma) and developed with One Step nitroblue tetrazolium–5-bromo-4-chloro-3-indolylphosphate (NBT-BCIP; Pierce Biotechnology, Inc., Rockford, IL) or ECL Plus reagent (GE Healthcare), respectively.

Techniques: Fractionation, Infection, Generated, Gradient Centrifugation, TCA Precipitation, SDS Page, Western Blot

Journal: Infection and Immunity

Article Title: Biochemical and Localization Analyses of Putative Type III Secretion Translocator Proteins CopB and CopB2 of Chlamydia trachomatis Reveal Significant Distinctions ▿

doi: 10.1128/IAI.00159-11

Figure Lengend Snippet: CopB and CopB2 partition differently in Triton X-114 extracts. Integral membrane association was tested by extracting C. trachomatis L2-infected HeLa cultures with Triton X-114 and separating proteins into aqueous and detergent soluble fractions. (A) Cultures were harvested at 20 h postinfection, and a parallel, mock-treated HeLa culture was harvested as a specificity control. Equivalent quantities of total protein from aqueous (Aq)- and detergent (Det)-soluble samples were resolved by SDS-PAGE in 12% polyacrylamide gels. Immunoblots were probed with anti-MOMP, anti-GroEL, anti-CopN, or anti-IncG as a fractionation control and with antibody specific for CopB or CopB2. Proteins were visualized by probing with secondary antibodies conjugated to alkaline phosphatase and development with NBT-BCIP. (B) HeLa cultures were mock treated or C. trachomatis L2 infected at an MOI of 10. Cultures were harvested at 2 h postinfection, extracted with Triton X-114, and probed by immunoblotting with the indicated antibodies. Proteins were visualized by probing with secondary antibodies conjugated to horseradish peroxidase and development with ECL Plus chemiluminescence reagent.

Article Snippet: After incubation with specific primary antibodies, immunoblots were probed with the appropriate alkaline phosphatase or horseradish peroxidase-conjugated secondary antibodies (Sigma) and developed with One Step nitroblue tetrazolium–5-bromo-4-chloro-3-indolylphosphate (NBT-BCIP; Pierce Biotechnology, Inc., Rockford, IL) or ECL Plus reagent (GE Healthcare), respectively.

Techniques: Infection, SDS Page, Western Blot, Fractionation

Journal: Proteome Science

Article Title: Proteomic analysis of primary duck hepatocytes infected with duck hepatitis B virus

doi: 10.1186/1477-5956-8-28

Figure Lengend Snippet: Confirmation of the differentially expressed proteins of annexin A2 and beta-actin by Western blot . The equal cell lysates of control and DHBV infected PDHs at 12, 24, 72 and 120 h post-infection were separated by SDS-PAGE, and detected by Western blotting with mouse anti-beta-actin and anti-duck annexin A2 as primary antibodies and then followed by horseradish peroxidase-conjugated goat anti-mouse IgG as secondary antibody (A). The same amount protein of each sample were applied to a parallel SDS-PAGE gel and stained with Coomassie brilliant blue (B).

Article Snippet: The blots were then washed four times with PBS containing 0.1% Tween-20, and incubated with the appropriate horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology) 1 hour at room temperature.

Techniques: Western Blot, Infection, SDS Page, Staining

Journal: PLoS ONE

Article Title: Cardiac-Oxidized Antigens Are Targets of Immune Recognition by Antibodies and Potential Molecular Determinants in Chagas Disease Pathogenesis

doi: 10.1371/journal.pone.0028449

Figure Lengend Snippet: Oxidative adducts are enhanced in experimental and human myocardium during Chagas disease. ( A ) Sprague-Dawley rats (or C3H/HeN mice) were infected with T. cruzi, and cells were harvested at day 40 (acute stage) and 180 (chronic stage) post-infection. Heart homogenates were resolved on 10% acrylamide gels, and Western blotting was performed with specific antibodies to detect 4 hydroxynonenal (4-HNE, panel a ), malondialdehyde (MDA, panel b ), dinitrophenyl (DNP)-derivatized carbonyl ( panel c ), and 3-nitrotyrosine (3NT, panel d ) adducts. Coomassie blue staining of membranes ( panel e ) confirmed the equal loading of samples. ( B ) Cryostat sections of human cardiac biopsies (5-µm) from normal healthy donors ( panels a, c, e ) and chagasic patients ( panels b, d, f ) were submitted to immunohistochemistry as described in Materials and Methods . Shown are representative images of immunostaining with anti-4-HNE antibodies ( panels a, b ). Tissue sections were incubated with DNPH to derivatize carbonyl proteins, and immunostaining was performed with anti-DNP antibody ( panels c–f ). HRP-conjugated ( panels a–d ) and rhodamine-conjugated ( panels e, f ) secondary antibodies were utilized to capture the color (brown) or fluorescence signal, respectively.

Article Snippet: Membranes were probed with sera from normal or chagasic rats, mice or human patients (1∶100 dilution) followed by HRP-conjugated secondary antibody (1∶5000, BioRad), and signal was detected by an ECL plus chemiluminiscence detection system (GE-Healthcare).

Techniques: Mouse Assay, Infection, Western Blot, Multiple Displacement Amplification, Staining, Immunohistochemistry, Immunostaining, Incubation, Fluorescence

Journal: Oncotarget

Article Title: Involvement of urokinase receptor in the cross-talk between human hematopoietic stem cells and bone marrow microenvironment

doi: 10.18632/oncotarget.11115

Figure Lengend Snippet: Full-length suPAR increases adhesion of KG1 cells to fibronectin Panel A: KG1 cells were resuspended in 1mg/ml BSA-RPMI at a density of 1×10 6 cells/ml, pre-incubated with or without suPAR (20 nM), uPAR 84-95 or scrambled peptide (10 nM) for 30′ at 37°C and plated on plastic-bound fibronectin for 16 h at 4°C. Attached cells were fixed with 3% PFA and stained with crystal violet; stain was eluted and its absorbance at 540 nm was measured by a spectrophotometer. (*) p≤0.05, as determined by the Student's t test. Panel B: suPAR-treated or untreated KG1 cells were fixed with PFA, incubated with antibodies against total or active β1 integrin or with nonimmune mouse Ig and, then, with FITC anti-mouse IgG. Cells were finally analyzed by flow cytometry using a FACScan (MFI: Mean Fluorescence Intensity). (*) p≤0.05, as determined by the Student's t test. Panel C: Cell surface antigens of suPAR-treated or untreated KG1 cells were biotinylated with EZ-link sulfo-NHS-LC-biotin. Cells were lysed; 50 μg of cell lysates were analyzed by Western blot with an antibody against total β1 integrin, 400 μg of cell lysates were incubated with an antibody against active β1 integrin or nonimmune mouse Ig. Immunocomplexes were then precipitated by protein A-Sepharose, eluted and analyzed by Western blot with horseradish peroxidase-conjugated streptavidin.

Article Snippet: Polyclonal anti-caspase3 and anti-VN antibodies, the monoclonal anti-PARP1 and anti-β1 integrin antibodies were purchased from Santa Cruz (Santa Cruz, CA); horseradish peroxidase-conjugated secondary antibodies from Bio-Rad (Hercules, CA) and FITC anti-mouse IgG from Jackson Lab. (West Grove, PA).

Techniques: Incubation, Staining, Spectrophotometry, Flow Cytometry, Cytometry, Fluorescence, Western Blot

Journal: PLoS ONE

Article Title: Serotonylation of Vascular Proteins Important to Contraction

doi: 10.1371/journal.pone.0005682

Figure Lengend Snippet: α-actin is serotonylated in aortic smooth muscle cells and inhibition of TG activity reduces aortic contraction to 5-HT. A. Immunoprecipitation of smooth muscle α-actin from rat aortic homogenates exposed to 5-HT-biotin in a standard transglutaminase reaction. Blots were developed using a streptavidin secondary (top), or exposed to a primary antibody against α-actin (bottom) and developed using standard horseradish peroxidase secondary antibody. Representative of N = 6 different experiments. B. Effect of vehicle (filled symbol) and cystamine (0.1–1 mM; open symbol) on 5-HT (top) and KCl (bottom)-induced contraction in isolated rat aorta. * indicates statistical difference from vehicle-incubated values. Points and vertical lines represent means±SEM for number of animals in parentheses.

Article Snippet: Samples were blocked overnight at 4°C in 4% chick egg ovalbulmin [TBS-0.1% Tween+0.025% NaN3, ], washed in TBS-Tween for 20 minutes, and incubated with streptavidin-linked, horseradish peroxidase-conjugated secondary antibody (1∶2000, 1 hr, 4°C GE Healthcare, Piscataway NJ, USA).

Techniques: Inhibition, Activity Assay, Immunoprecipitation, Isolation, Incubation

Journal: Journal of autoimmunity

Article Title: Mechanisms of Neuropsychiatric Lupus: The Relative Roles of the Blood-CSF versus Blood-Brain Barrier

doi: 10.1016/j.jaut.2018.03.001

Figure Lengend Snippet: Blood-brain barrier permeability is preserved in NPSLE mice Staining for endogenous intravascular proteins or following exogenous tracer challenges showed no indication of BBB hyper-permeability correlating with NPSLE progression. A. Female MRL/lpr mice (at 16 weeks of age, when active systemic and NPSLE manifestations are prevalent) and age-matched congenic control MRL/+ mice were co-stained for CD31 and for mouse IgG. B. Quantification shows that the prevalence of infrequent leakage incidents did not significantly differ between genotypes (P > 0.9 as determined by a non-parametric Mann-Whitney U test). Leakage was defined as present when IgG was localized outside the endothelial area (outlined by the CD31 staining). At least four non-sequential sagittal sections per animal were scanned under the microscope, and analyzed by an individual blinded to the animal genotype. C. MRL/lpr and control MRL/+ mice (at 16 weeks of age) were injected with 10 kDa rhodamine-dextran for short-term (5–10 min) tracer challenges and co-stained for CD31. D. Quantification shows that the prevalence of rare leakage incidents did not significantly differ between genotypes (P > 0.9 as determined by a non-parametric Mann-Whitney U test). In both assays (A and C), fluorescence imaging was performed on cortical sagittal sections (covering visual and somatosensory regions of the cerebral cortex). BBB function of the overall vascular network was generally preserved, and the tracer signal restricted to the vessel lumen (upper panels). In both lupus and control mice, tracer leakage was relatively uncommon, and mostly restricted to parenchymal tissue surrounding medium-large size vessels (arrows, insets in lower panels). Images from MRL/lpr mice are representative of both genotypes. N=5 mice per group; for quantifications 10–24 sections per mouse were evaluated, scale bars 50 µm upper panels 100 µm lower panels. E. TEM imaging of cortical capillaries from HRP injected, 16 week old female MRL/lpr mice. Capillary lumen is filled with HRP. No tracer leakage found demonstrating a functional tight junction (arrow) and no evidence of transcytosis. N=5 mice; scale bar 0.5 µm. All data are mean ± s.e.m.

Article Snippet: HRP: Deeply anesthetized 16-week old MRL/lpr mice were injected with 0.4 mL of HRP (Sigma Aldrich, HRP type II, 10 mg/20 g mouse body weight, dissolved in PBS) through the tail vein.

Techniques: Permeability, Mouse Assay, Staining, MANN-WHITNEY, Microscopy, Injection, Fluorescence, Imaging, Transmission Electron Microscopy, Functional Assay

Journal: Journal of autoimmunity

Article Title: Mechanisms of Neuropsychiatric Lupus: The Relative Roles of the Blood-CSF versus Blood-Brain Barrier

doi: 10.1016/j.jaut.2018.03.001

Figure Lengend Snippet: BCSFB dysfunction in NPSLE mice points to an alternative route of antibody entry into the CNS ). B. Confocal images of 10 µm sagittal brain sections of 16 week old MRL/lpr mice stained for endogenous IgG (green) and nuclei (DAPI, blue). As expected, IgG is found in CP vessels (asterisks). IgG is also found at the apical side of the epithelial barrier only in 16 week old MRL/lpr mice (lower panels, arrow heads) but not in control MRL/+ mice (upper panel). Ventricular BCSFB IgG staining is more abundant in CP regions where immune cells infiltrated the stroma (highly infiltrated regions, lower panel. less infiltrated regions, middle panel). Scale bars 20 µm and 5 µm in insets. C. TEM imaging of 16 week old MRL/lpr CP stained with IgG-HRP. IgG is localized at the ventricular surface of the BCSFB, covering epithelial cells microvilli (left panel, arrows in inset). Abundant IgG deposits are also found in the basolateral space between epithelial cells (right panel, arrows), reaching the epithelial tight junction (right panel, arrow head in inset). Scale bars 2 µm, and 1 µm in insets.

Article Snippet: HRP: Deeply anesthetized 16-week old MRL/lpr mice were injected with 0.4 mL of HRP (Sigma Aldrich, HRP type II, 10 mg/20 g mouse body weight, dissolved in PBS) through the tail vein.

Techniques: Mouse Assay, Staining, Transmission Electron Microscopy, Imaging

Journal: Redox Biology

Article Title: Peperomin E and its orally bioavailable analog induce oxidative stress-mediated apoptosis of acute myeloid leukemia progenitor cells by targeting thioredoxin reductase

doi: 10.1016/j.redox.2019.101153

Figure Lengend Snippet: PepE target the TrxR1 through binding its Sec residue. (A) Inhibition assay of purified recombinant human TrxR1 or TrxR1 without Sec498 residue (nSec498, 0.1 μM) by PepE. The NADPH-reduced proteins were incubated with the indicated concentrations of PepE, and the enzyme's activity was determined by the DTNB assay (Data are presented as mean ± SD, n = 6). (B) Molecular docking of PepE with TrxR1 was carried out using the covalent docking protocol in the Schrodinger Suite. The yellow line indicated the covalent carbon bond, the green dotted lines indicate the hydrogen bonds. (C) Comparison of the binding activity between PepE and TrxR1 protein (red line), PepE and TrxR1 nSec498 protein (blue line), and PepA and TrxR1 protein (black line) through BLI analysis. (D) Kinetic analysis of the interaction between NADPH reduced TrxR1 (0.9 μM) or NADPH reduced TrxR1 nSec498 (0.9 μM) protein and PepE by BLI. The Super Streptavidin (SSA) biosensor tips coated with proteins (0.9 μM) were dipped in increasing concentrations of PepE (0.625, 1.25, 2.5, 5 and 10 μM) to measure the binding affinity of PepE to the proteins (K on ) and subsequently moved to wells containing buffer to measu re dissociation rates (K dis ). The affinity constant (K D ) was calculated as the ratio of the K dis to the K on. (E) Kinetic analysis of the interaction between TrxR1 (0.9 μM) and different concentrations of PepA (1.25, 2.5, 5, 10 and 20 μM) by BLI. (F) The interaction of the PepE with the Sec residue in the C -terminal active center of TrxR1. HRP-conjugated streptavidin (HRP-Strep) detection of BIAM labeling of free Selenol in TrxR1 enzyme at pH 6.5 after this enzyme was incubated with different concentrations of PepE (1, 5 and 10 μM), DMSO(negative control), and DNCB (positive control, 5 μM), ** P

Article Snippet: Horseradish peroxidase (HRP)-conjugated secondary antibody (goat anti-rabbit), human TrxR1 shRNA plasmids, control shRNA plasmids, TrxR1 CRISPR activation plasmids, control CRISPR activation plasmids were obtained from Santa Cruz Biotech.

Techniques: Binding Assay, Size-exclusion Chromatography, Inhibition, Purification, Recombinant, Incubation, Activity Assay, DTNB Assay, Labeling, Positive Control

Journal: Oncotarget

Article Title: Minimal contribution of ERK1/2-MAPK signalling towards the maintenance of oncogenic GNAQQ209P-driven uveal melanomas in zebrafish

doi: 10.18632/oncotarget.9207

Figure Lengend Snippet: Oncogenic GNAQ Q209P -mediated activation of ERK and YAP signalling in choroidal melanocytes at the junction between RPE and choroid A-D. Transverse sections of formalin-fixed and paraffin-embedded eye tissues of F 1 generation, 5-month-old Tg ( mitfa :GNAQ Q209P ) zebrafish. (A) H E staining demonstrating choroidal hyperplasia (black arrowheads). White dashed box indicates the region of the choroid magnified in B-D. (B-D) Transverse sections of formalin-fixed and paraffin-embedded eye tissues were stained by IHC, visualized by ImmPact NovaRed peroxidase (HRP) substrate then counterstained with hematoxylin (blue). (B) Negative control: section incubated with 1x PBS instead of primary antibody. (C) Immunoreactivity to pERK1/2 (read-out of ERK activation; black arrowheads) in melanocytes at the interface between the RPE and choroid. (D) YAP-positive nuclei (read-out of YAP activation; black arrowheads) in the same cells. Scale bars, 20 μm.

Article Snippet: To remove excess unbound primary antibody, membranes were washed three times with 1x TBST buffer then incubated with an appropriate horseradish peroxidase (HRP)-conjugated secondary antibody (Cell Signalling Technology, 1:1000 dilution) for 1 hour at room temperature.

Techniques: Activation Assay, Staining, Immunohistochemistry, Negative Control, Incubation

Journal: PLoS Pathogens

Article Title: A Role for the SmpB-SsrA System in Yersinia pseudotuberculosis Pathogenesis

doi: 10.1371/journal.ppat.0020006

Figure Lengend Snippet: SmpB-SsrA Mediated Protein Tagging Activity Is Observed in Y. pseudotuberculosis (A) Schematic representation of the λ-cI-N-trpAt reporter construct encoded on the pKW540 plasmid and anticipated outcomes of protein tagging in wild-type (WT) or ΔBA strains. (B) The λ-cI-N-trpAt reporter was induced in wild-type and mutant (ΔBA) strains, and protein samples were analyzed by Western blot using HRP-conjugated anti-H6 serum. MW stds, protein molecular weight standards.

Article Snippet: Resolved proteins were transferred to polyvinylidene difluoride (PVDF) membrane and probed with horseradish peroxidase (HRP) conjugated anti-H6 antibodies (Santa Cruz Biotechnology, Santa Cruz, California, United States) to detect the H6 epitope present in the λ-cI-N protein.

Techniques: Activity Assay, Construct, Plasmid Preparation, Mutagenesis, Western Blot, Molecular Weight