bovine serum albumin bsa Search Results


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  • 99
    Vector Laboratories bovine serum albumin
    Bovine Serum Albumin, supplied by Vector Laboratories, used in various techniques. Bioz Stars score: 99/100, based on 1633 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher bsa
    GAL efficiently labels Gal and GalNAc residues on cell surface glycoproteins. ( A ) Asialo-K20 cells were incubated in <t>PBS</t> with 5 mg/mL <t>BSA,</t> pH 6.7, containing 250 μM aminooxy-biotin in the presence or absence of 10 mM aniline and 50 U/mL galactose
    Bsa, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 27155 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore bsa
    Selected bacteria reside in LC3-positive phagolysosomes.A and B. Long-term infected <t>(LTI)</t> macrophages for at least 2 years and recently infected (RI) with BCG-GFP ancestral strain for 24 h were stained with Lysotracker (A, left panel) or <t>DQ-BSA</t> (B, right panel).C and D. Quantitative analysis of the fluorescence intensity of Lysotracker (C) and DQ-BSA (D) associated with phagosomes in LTI macrophages compared with RI cells. Around 80 phagosomes were quantified in Lysotracker or DQ-BSA stained macrophages from three independent experiments.E and F. Recently infected macrophages (RI) during 24 h (E) and LTI (F) macrophages (2 years old) were subjected to immunofluorescence and endogenous LC3 and LAMP-2 were detected using specific antibodies. Insets depict colocalization with single markers.G and H. Quantitative analysis of the fluorescence intensity of LC3 (G) and LAMP-2 (H) associated with phagosomes of RI and LTI macrophages. Around 130 phagosomes were quantified for LC3 detection and 190 phagosomes for LAMP-2 detection from three independent experiments.Data represent the mean ± S.E.M., (**) P ≤ 0.01, (***) P ≤ 0.001 and (****) P ≤ 0.0001 from two-tailed Student's t -test. Nuclei were stained with Hoechst 33258. Scale bars: 10 μm.
    Bsa, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 43463 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore bovine serum albumin bsa
    Selectivity evaluation of the biosensor for the detection of thrombin against other proteins of Lysozyme, Trypsase, <t>lgG,</t> <t>BSA,</t> and HSA. The concentration of the thrombin was 0.2 µM. However, the concentration of Lysozyme, Trypsase, lgG, BSA, and HSA was 2 µM. No thrombin or other proteins were used in the blank group. Inset: the changes in fluorescence intensity (F/F 0 ), F and F 0 , are the fluorescence intensities in the presence and absence of protein, respectively. The error bar was calculated in three independent experiments.
    Bovine Serum Albumin Bsa, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 32017 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    bsa  (Abcam)
    99
    Abcam bsa
    The ARC CARD is required for the ability of ARC to suppress TNF α -induced necrosis. ( a ) Immunoblot showing inhibition of TNF α -induced <t>HMGB1</t> release by wild-type ARC but not by the CARD-defective double-point ARC mutant (L31F; G69R). L929 cells were stably transduced with empty vector (Φ), ARC-HA (ARC) or ARC-HA double-point CARD mutant (DM). Immunblot of bovine serum albumin <t>(BSA)</t> in media used as loading control. ( b ) Inhibition of TNF α -induced LDH release by ARC requires the CARD. Data shown as mean±S.E. from n =4. *** P -value
    Bsa, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 11355 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher bovine serum
    The ARC CARD is required for the ability of ARC to suppress TNF α -induced necrosis. ( a ) Immunoblot showing inhibition of TNF α -induced <t>HMGB1</t> release by wild-type ARC but not by the CARD-defective double-point ARC mutant (L31F; G69R). L929 cells were stably transduced with empty vector (Φ), ARC-HA (ARC) or ARC-HA double-point CARD mutant (DM). Immunblot of bovine serum albumin <t>(BSA)</t> in media used as loading control. ( b ) Inhibition of TNF α -induced LDH release by ARC requires the CARD. Data shown as mean±S.E. from n =4. *** P -value
    Bovine Serum, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 8779 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Jackson Immuno bsa
    <t>GDF11-induced</t> phenotypes require SMAD4 and ID2 (A) SMAD4 knockdown blocks the ability of GDF11 to inhibit 3D invasion in MDA-MB-231 cells. shLuc was induced as a control shRNA. (B) Inducible reconstitution of SMAD4 in SMAD4-null MDA-MB-468 basal-like TNBC cells. FLAG-LacZ was induced as a control overexpression. MCF10A-5E cell extracts were included to gauge endogenous abundance of SMAD4. (C) SMAD4 reconstitution is sufficient to confer GDF11 responsiveness to MDA-MB-468 cells. Control cells express inducible FLAG-LacZ. (D) Differential transcriptomic profiling of GDF11- and TGFβ-stimulated NMuMG 3D cultures reveals Id2 as a candidate GDF11 effector. NMuMG spheroids were grown for 6 days in culture and stimulated with either cytokine for 4 hours and RNA expression was measured by Illumina BeadChip arrays. Representative data are shown from n = 4 biological replicates. (E) Knockdown of Id2 blocks the ability of GDF11 to promote 3D rounding in NMuMG cells. shGFP was induced as a control shRNA. (F) ID2 knockdown blocks the ability of GDF11 to inhibit 3D invasion in MDA-MB-231 cells. shLuc was induced as a control shRNA. (G) Basal-like breast cancer (BLBC) patients with GDF11 expression ( > 1.5 copies per cell) and elevated ACVR2B and TGFBR3 show increased abundance of ID2 ). (H) Tumor bioluminescence of MDA-MB-231 intraductal xenografts co-inoculated with 200 ng GDF11 or <t>BSA</t> control. Tumor bioluminescence was measured at day 2, and every seven days post inoculation. (I and J) Recombinant GDF11 reduces intraductal proliferation but does not affect apoptosis. Intraductal lesions were stained for Ki67 (I) or cleaved caspase-3 (ClvC3), and positive cells per 10× field of view (FOV) were quantified for paired GDF11 and control glands (J). (K) ID2 knockdown blocks the ability of GDF11 to suppress intraductal growth of MDA-MB-231 cells. shLacZ was induced as a control shRNA with shID2 #2 at day 10. Data are shown as the mean ± SEM of n = 4 (A, C, E, F) biological replicates or 10 mice (K) or the median ± 90% nonparametric confidence interval of n ). 3D cultures were assessed at day 16 (A, F), 12 (C), and 20 (E). Scale bar is 200 μm (A, C, F) and 40 μm (I). n.s., not significant ( p > 0.05 by two-sided Ward’s test [A, F] or sign-rank test [J]). .
    Bsa, supplied by Jackson Immuno, used in various techniques. Bioz Stars score: 99/100, based on 2796 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    GE Healthcare bsa
    Purification of <t>anti-BSA-digoxin</t> bispecific antibodies. Anti-BSA-digoxin bispecific antibodies were purified from the serum using a series of purification methods. IgG fraction was obtained using 50% saturated ammonium sulfate precipitation, followed by passing through a protein A column. Bispecific antibody was further purified by affinity chromatography, using <t>digoxin-Sepharose</t> and BSA-Sepharose, after which a pure antibody fraction possessing bispecificity was obtained. Monospecific and bispecific antibody ELISAs were performed to monitor antibody titers during the entire purification process and the results were plotted.
    Bsa, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 99/100, based on 2411 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam bovine serum albumin bsa
    Cell polarity disrupts the cis interaction between P2Y 2 R and β 3 integrin and infectivity. (A) Confocal image of polarized Vero E6 cells expressing antibody-stained basolateral α v β 3 and apical P2Y 2 R. The top panel is a confocal image in which the focus plane was parallel to the monolayer ( XY scan), whereas the bottom panel shows the focus plane as a vertical cross section of the monolayer ( XZ scan). The white line indicated by the arrow in the XY scan indicates the path of the XZ scan. To enable visualization, Vero E6 cells were transiently transfected with P2Y 2 R and plated at confluence in eight-well Nunc Lab-Tek chambers and allowed to propagate for 1–2 d. At room temperature, cells were fixed with 3% paraformaldehyde for 20 min and subsequently permeabilized with 0.2% Triton X-100 (in <t>PBS)</t> for 15 min. Cells were blocked for nonspecific staining with 1% <t>BSA</t> in PBS for 30 min and then incubated with mouse monoclonal anti-integrin α v β 3 (1:40, Millipore MsxHu, MAB#1976) and rabbit polyclonal anti-P2Y 2 (1:100, Abcam, ab10270) were used with Alexa 488 and Alexa 580, respectively. (B) Plot of SNV FFU/ml vs. cells in suspension and on plates. CHO-A24, TIME, and Vero E6 cells were plated at confluence in vitronectin treated 48-well plates for 24 h. Confluent cells in triplicate wells were inoculated with 0.1 moi SNV for 1 h, washed in low pH media, and then incubated in normal media for 24 h. Media from infected cells was collected after 24 h diluted and subjected to a 7-day focus assay in Vero cells (see Supplemental Figure S1E for typical SNV FFU). ** p
    Bovine Serum Albumin Bsa, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 4867 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc bovine serum albumin
    Cell polarity disrupts the cis interaction between P2Y 2 R and β 3 integrin and infectivity. (A) Confocal image of polarized Vero E6 cells expressing antibody-stained basolateral α v β 3 and apical P2Y 2 R. The top panel is a confocal image in which the focus plane was parallel to the monolayer ( XY scan), whereas the bottom panel shows the focus plane as a vertical cross section of the monolayer ( XZ scan). The white line indicated by the arrow in the XY scan indicates the path of the XZ scan. To enable visualization, Vero E6 cells were transiently transfected with P2Y 2 R and plated at confluence in eight-well Nunc Lab-Tek chambers and allowed to propagate for 1–2 d. At room temperature, cells were fixed with 3% paraformaldehyde for 20 min and subsequently permeabilized with 0.2% Triton X-100 (in <t>PBS)</t> for 15 min. Cells were blocked for nonspecific staining with 1% <t>BSA</t> in PBS for 30 min and then incubated with mouse monoclonal anti-integrin α v β 3 (1:40, Millipore MsxHu, MAB#1976) and rabbit polyclonal anti-P2Y 2 (1:100, Abcam, ab10270) were used with Alexa 488 and Alexa 580, respectively. (B) Plot of SNV FFU/ml vs. cells in suspension and on plates. CHO-A24, TIME, and Vero E6 cells were plated at confluence in vitronectin treated 48-well plates for 24 h. Confluent cells in triplicate wells were inoculated with 0.1 moi SNV for 1 h, washed in low pH media, and then incubated in normal media for 24 h. Media from infected cells was collected after 24 h diluted and subjected to a 7-day focus assay in Vero cells (see Supplemental Figure S1E for typical SNV FFU). ** p
    Bovine Serum Albumin, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 5298 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Santa Cruz Biotechnology bovine serum albumin bsa
    ). (D) ELISA sugar-binding assay for <t>PdCoV</t> S1-NTD. Here, the ELISA plates were precoated with sugar-rich mucin, and then PdCoV S1-NTD was added and incubated with mucin. Mucin-bound S1-NTD was detected using antibodies recognizing its C-terminal His 6 tag. Porcine epidemic diarrhea virus (PEDV) S1 was used as the positive control. PdCoV S1-CTD, SARS-CoV S1-CTD, and <t>BSA</t> were used as negative controls. A plate without mucin was used as an additional negative control. Statistical analyses were performed using two-tailed t test. Error bars indicate standard errors of the means (SEM) ( n = 5). ***, P
    Bovine Serum Albumin Bsa, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 94/100, based on 4064 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad bovine serum albumin bsa
    Preferential binding of <t>PvNod41</t> to denatured proteins . (A) PvNod41 binding assay. Purified PvNod41 was incubated with either native (N) or denatured (D) proteins pre-immobilized on agarose-beads. After incubation, samples were extensively washed with PBS. PvNod41 that was bound to immobilized proteins on the matrix was recovered by boiling the sample with Laemmli buffer and analyzed by 12% SDS-PAGE and Coomassie Brilliant Blue staining. <t>BSA,</t> Bovine Serum Albumin; α2M, α2-Macroglobulin. (B) Far western blot assay. Bovine serum albumin (BSA) and casein, either native or denatured by boiling were blotted onto nitrocellulose, probed with purified PvNod41, and immunodetected with anti-PvNod41 antiserum as described in the Methods section.
    Bovine Serum Albumin Bsa, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 93/100, based on 2075 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson bovine serum albumin bsa
    Mouse hippocampal and cortical neurons (visualized by labeling for PGP 9.5) and nuclei of all cells (visualized by DAPI labeling) after 3 days culture on <t>BSA</t> (A, B, C, and D) merosin (E, F, G, and H) and FN (I, J, K, and L). Lengths of neurites (including axons) are greater on FN than BSA or merosin. Scale bar = 100 μm.
    Bovine Serum Albumin Bsa, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 94/100, based on 1822 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore bovine serum albumin
    Mouse hippocampal and cortical neurons (visualized by labeling for PGP 9.5) and nuclei of all cells (visualized by DAPI labeling) after 3 days culture on <t>BSA</t> (A, B, C, and D) merosin (E, F, G, and H) and FN (I, J, K, and L). Lengths of neurites (including axons) are greater on FN than BSA or merosin. Scale bar = 100 μm.
    Bovine Serum Albumin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 96597 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    GAL efficiently labels Gal and GalNAc residues on cell surface glycoproteins. ( A ) Asialo-K20 cells were incubated in PBS with 5 mg/mL BSA, pH 6.7, containing 250 μM aminooxy-biotin in the presence or absence of 10 mM aniline and 50 U/mL galactose

    Journal: Glycobiology

    Article Title: Glycoproteomics enabled by tagging sialic acid- or galactose-terminated glycans

    doi: 10.1093/glycob/cws144

    Figure Lengend Snippet: GAL efficiently labels Gal and GalNAc residues on cell surface glycoproteins. ( A ) Asialo-K20 cells were incubated in PBS with 5 mg/mL BSA, pH 6.7, containing 250 μM aminooxy-biotin in the presence or absence of 10 mM aniline and 50 U/mL galactose

    Article Snippet: For the oxime ligation step, cells were suspended to 1 × 107 cells/mL in PBS/5% BSA, pH 6.7 containing 250 μM aminooxy-biotin, aminooxy-AF488 (Invitrogen Corporation) or aminooxy-FLAG peptide and 10 mM aniline (Sigma–Aldrich) for 90 min at 4°C with end-over-end mixing.

    Techniques: Incubation

    Validation of methylation on AKT1 at lysine 14 by specific antibody A. The enzyme-linked immunosorbent assay of the anti-monomethyl AKT1 (Lys 14) antibody. A rabbit was immunized with synthetic peptides, including Lys 14 monomethylation, and the affinity purification was carried out against the methylated synthetic peptides. The original serum (S) and the flow through (FT) show equal reactivity against the carrier protein. The purified antibody (PA) shows a stronger reactivity against the methylated peptide than the serum (S). B. Testing of specific antibody (SA) and non-specific antibody (NS) against the unmodified peptide by enzyme-linked immunosorbent assay. C. Validation of the anti-K14 monomethylated AKT1 antibody. Recombinant AKT1 protein or BSA and S-adenosyl-L-methionine were incubated in the presence or absence of recombinant SMYD3, and the reaction products were analyzed by SDS-PAGE, followed by western blot analysis using the anti-K14 monomethylated AKT1 antibody. The nitrocellulose membrane was stained with MemCode™ Reversible Stain Kit after western blot analysis. D. 293T cells were co-transfected with Mock, FLAG-AKT1-WT, FLAG-AKT1-K14A or FLAG-AKT1-K14R together with a wild-type SMYD3 expression vector (pcDNA3.1-SMYD3-WT) or an enzyme-inactive SMYD3 expression (pcDNA3.1-SMYD3-ΔEEL). After 48 hours of incubation, cells were treated with 100 ng/ml of EGF for 5 min, and then lysed with CelLytic™ M mammalian cell lysis/extraction reagent containing a protease inhibitor cocktail and a phosphatase cocktail. The samples were immunoblotted with anti-K14 monomethylated AKT1, anti-phospho AKT (Thr 308) and anti-FLAG antibodies after immunoprecipitating with anti-FLAG ® M2 affinity gel.

    Journal: Oncotarget

    Article Title: SMYD3-mediated lysine methylation in the PH domain is critical for activation of AKT1

    doi: 10.18632/oncotarget.11898

    Figure Lengend Snippet: Validation of methylation on AKT1 at lysine 14 by specific antibody A. The enzyme-linked immunosorbent assay of the anti-monomethyl AKT1 (Lys 14) antibody. A rabbit was immunized with synthetic peptides, including Lys 14 monomethylation, and the affinity purification was carried out against the methylated synthetic peptides. The original serum (S) and the flow through (FT) show equal reactivity against the carrier protein. The purified antibody (PA) shows a stronger reactivity against the methylated peptide than the serum (S). B. Testing of specific antibody (SA) and non-specific antibody (NS) against the unmodified peptide by enzyme-linked immunosorbent assay. C. Validation of the anti-K14 monomethylated AKT1 antibody. Recombinant AKT1 protein or BSA and S-adenosyl-L-methionine were incubated in the presence or absence of recombinant SMYD3, and the reaction products were analyzed by SDS-PAGE, followed by western blot analysis using the anti-K14 monomethylated AKT1 antibody. The nitrocellulose membrane was stained with MemCode™ Reversible Stain Kit after western blot analysis. D. 293T cells were co-transfected with Mock, FLAG-AKT1-WT, FLAG-AKT1-K14A or FLAG-AKT1-K14R together with a wild-type SMYD3 expression vector (pcDNA3.1-SMYD3-WT) or an enzyme-inactive SMYD3 expression (pcDNA3.1-SMYD3-ΔEEL). After 48 hours of incubation, cells were treated with 100 ng/ml of EGF for 5 min, and then lysed with CelLytic™ M mammalian cell lysis/extraction reagent containing a protease inhibitor cocktail and a phosphatase cocktail. The samples were immunoblotted with anti-K14 monomethylated AKT1, anti-phospho AKT (Thr 308) and anti-FLAG antibodies after immunoprecipitating with anti-FLAG ® M2 affinity gel.

    Article Snippet: Mass spectrometry AKT1 samples reacted with bovine serum albumin (BSA) or SMYD3 in vitro and immunoprecipitated AKT1 samples from 293T cells and HeLa cells were separated on SDS-PAGE and stained with Simply Blue Safe Stain (Thermo Fisher Scientific).

    Techniques: Methylation, Enzyme-linked Immunosorbent Assay, Affinity Purification, Flow Cytometry, Purification, Peptide ELISA, Recombinant, Incubation, SDS Page, Western Blot, Staining, Transfection, Expressing, Plasmid Preparation, Lysis, Protease Inhibitor

    Confocal images of TRPV1 (A–D) and SP immunolabeling (E–H) expression in (A, E) BA, (B, F) AICA, (C, G) SMA, and (D, H) RA. Arrows indicate labeled: longitudinal (a), circumferential (b) fibers. Insets in A–D, show negligible labeling in BA, AICA, SMA, and RA incubated with antigen-adsorbed TRPV1 antibodies and secondary antibodies. Insets in E–H show negligible labeling in the BA, AICA, SMA, and radiating arterioles incubated with 1% BSA-PBS and secondary antibodies. Scale bars in microns.

    Journal: Neuroscience

    Article Title: CO-LOCALIZATION OF THE VANILLOID CAPSAICIN RECEPTOR AND SUBSTANCE P IN SENSORY NERVE FIBERS INNERVATING COCHLEAR AND VERTEBRO-BASILAR ARTERIES

    doi: 10.1016/j.neuroscience.2003.12.030

    Figure Lengend Snippet: Confocal images of TRPV1 (A–D) and SP immunolabeling (E–H) expression in (A, E) BA, (B, F) AICA, (C, G) SMA, and (D, H) RA. Arrows indicate labeled: longitudinal (a), circumferential (b) fibers. Insets in A–D, show negligible labeling in BA, AICA, SMA, and RA incubated with antigen-adsorbed TRPV1 antibodies and secondary antibodies. Insets in E–H show negligible labeling in the BA, AICA, SMA, and radiating arterioles incubated with 1% BSA-PBS and secondary antibodies. Scale bars in microns.

    Article Snippet: The tissues were washed in fresh 0.02 M PBS (pH 7.4), permeabilized in 0.5% Triton-X 100 (Sigma, St. Louis, MO, USA) in 0.02 M PBS (pH 7.3) for 30 min, and immunoblocked in 10% goat serum in 1% bovine serum albumin (BSA) in 0.02 M PBS (1% BSA-PBS) for 60 min. For immunocytochemistry, tissues were incubated with rabbit anti-TRPV1 antiserum (diluted 1:1000 in 1% BSA-PBS; gift from Dr. Michael J Caterina, John Hopkins University) for 48 h. After washing in 1% BSA-PBS, tissues were subsequently incubated in Alexa-488-conjugated goat anti-rabbit IgG for 3 h (Molecular Probes; 1:100 in BSA-PBS).

    Techniques: Immunolabeling, Expressing, Labeling, Incubation

    Confocal images of the DRG double-immunolabeled for TRPV1 (A), and for SP (B). (C) A subset of neuronal cell bodies co-express TRPV1 and SP (white, arrows) in the merged image. Inset in (A) shows negligible labeling in the DRG when incubated with antigen-adsorbed TRPV1 antibodies and secondary antibodies. Inset in B shows negligible labeling in the DRG when incubated with 1% BSA-PBS and secondary antibodies. Scale bar in microns.

    Journal: Neuroscience

    Article Title: CO-LOCALIZATION OF THE VANILLOID CAPSAICIN RECEPTOR AND SUBSTANCE P IN SENSORY NERVE FIBERS INNERVATING COCHLEAR AND VERTEBRO-BASILAR ARTERIES

    doi: 10.1016/j.neuroscience.2003.12.030

    Figure Lengend Snippet: Confocal images of the DRG double-immunolabeled for TRPV1 (A), and for SP (B). (C) A subset of neuronal cell bodies co-express TRPV1 and SP (white, arrows) in the merged image. Inset in (A) shows negligible labeling in the DRG when incubated with antigen-adsorbed TRPV1 antibodies and secondary antibodies. Inset in B shows negligible labeling in the DRG when incubated with 1% BSA-PBS and secondary antibodies. Scale bar in microns.

    Article Snippet: The tissues were washed in fresh 0.02 M PBS (pH 7.4), permeabilized in 0.5% Triton-X 100 (Sigma, St. Louis, MO, USA) in 0.02 M PBS (pH 7.3) for 30 min, and immunoblocked in 10% goat serum in 1% bovine serum albumin (BSA) in 0.02 M PBS (1% BSA-PBS) for 60 min. For immunocytochemistry, tissues were incubated with rabbit anti-TRPV1 antiserum (diluted 1:1000 in 1% BSA-PBS; gift from Dr. Michael J Caterina, John Hopkins University) for 48 h. After washing in 1% BSA-PBS, tissues were subsequently incubated in Alexa-488-conjugated goat anti-rabbit IgG for 3 h (Molecular Probes; 1:100 in BSA-PBS).

    Techniques: Immunolabeling, Labeling, Incubation

    Adherence of rHX1 and rHX2 to HEp-2 cells. HEp-2 cells were incubated with (A) rHX2, (B) rHX1, and (C) PBS-BSA (negative control), and after washing, cells were incubated with rabbit antiserum to rHX2, antiserum to rHX1 and PBS-BSA, respectively. Then, HEp-2 cells were labeled with anti-rabbit IgG-FITC and examined by fluorescence microscopy.

    Journal: PLoS ONE

    Article Title: Pre-Absorbed Immunoproteomics: A Novel Method for the Detection of Streptococcus suis Surface Proteins

    doi: 10.1371/journal.pone.0021234

    Figure Lengend Snippet: Adherence of rHX1 and rHX2 to HEp-2 cells. HEp-2 cells were incubated with (A) rHX2, (B) rHX1, and (C) PBS-BSA (negative control), and after washing, cells were incubated with rabbit antiserum to rHX2, antiserum to rHX1 and PBS-BSA, respectively. Then, HEp-2 cells were labeled with anti-rabbit IgG-FITC and examined by fluorescence microscopy.

    Article Snippet: To each well was added 30 mM aminoacetic acid for 5 min, followed by 0.1% Triton X-100 (Sigma) for a further 5 min. After three washes with PBS, the wells were blocked with 3% PBS-BSA (GIBCO) overnight at 4°C, and then washed prior to use.

    Techniques: Incubation, Negative Control, Labeling, Fluorescence, Microscopy

    Selected bacteria reside in LC3-positive phagolysosomes.A and B. Long-term infected (LTI) macrophages for at least 2 years and recently infected (RI) with BCG-GFP ancestral strain for 24 h were stained with Lysotracker (A, left panel) or DQ-BSA (B, right panel).C and D. Quantitative analysis of the fluorescence intensity of Lysotracker (C) and DQ-BSA (D) associated with phagosomes in LTI macrophages compared with RI cells. Around 80 phagosomes were quantified in Lysotracker or DQ-BSA stained macrophages from three independent experiments.E and F. Recently infected macrophages (RI) during 24 h (E) and LTI (F) macrophages (2 years old) were subjected to immunofluorescence and endogenous LC3 and LAMP-2 were detected using specific antibodies. Insets depict colocalization with single markers.G and H. Quantitative analysis of the fluorescence intensity of LC3 (G) and LAMP-2 (H) associated with phagosomes of RI and LTI macrophages. Around 130 phagosomes were quantified for LC3 detection and 190 phagosomes for LAMP-2 detection from three independent experiments.Data represent the mean ± S.E.M., (**) P ≤ 0.01, (***) P ≤ 0.001 and (****) P ≤ 0.0001 from two-tailed Student's t -test. Nuclei were stained with Hoechst 33258. Scale bars: 10 μm.

    Journal: Cellular Microbiology

    Article Title: Experimental selection of long-term intracellular mycobacteria

    doi: 10.1111/cmi.12303

    Figure Lengend Snippet: Selected bacteria reside in LC3-positive phagolysosomes.A and B. Long-term infected (LTI) macrophages for at least 2 years and recently infected (RI) with BCG-GFP ancestral strain for 24 h were stained with Lysotracker (A, left panel) or DQ-BSA (B, right panel).C and D. Quantitative analysis of the fluorescence intensity of Lysotracker (C) and DQ-BSA (D) associated with phagosomes in LTI macrophages compared with RI cells. Around 80 phagosomes were quantified in Lysotracker or DQ-BSA stained macrophages from three independent experiments.E and F. Recently infected macrophages (RI) during 24 h (E) and LTI (F) macrophages (2 years old) were subjected to immunofluorescence and endogenous LC3 and LAMP-2 were detected using specific antibodies. Insets depict colocalization with single markers.G and H. Quantitative analysis of the fluorescence intensity of LC3 (G) and LAMP-2 (H) associated with phagosomes of RI and LTI macrophages. Around 130 phagosomes were quantified for LC3 detection and 190 phagosomes for LAMP-2 detection from three independent experiments.Data represent the mean ± S.E.M., (**) P ≤ 0.01, (***) P ≤ 0.001 and (****) P ≤ 0.0001 from two-tailed Student's t -test. Nuclei were stained with Hoechst 33258. Scale bars: 10 μm.

    Article Snippet: To analyse cell death, LTI or RI macrophages were scraped and resuspended in PBS with 1% BSA (Albumin bovine serum, Sigma, Germany).

    Techniques: Infection, Staining, Fluorescence, Immunofluorescence, Two Tailed Test

    Modulation of S1P homeostasis of K562 cells by sodium butyrate-induced differentiation into erythroblast-like cells. K562 cells were treated with 2 mM sodium butyrate (NaB); 72 hours later, we investigated the modulation of S1P homeostasis. (A) The mRNA levels of GYPA and ALAS were determined using real-time PCR. GAPDH was utilized as an internal control (n = 8/group). (B, C) The expression of CD235a was investigated with a flow cytometer (n = 3/group). (D) C 17 S1P formation assay. NaB-treated or vehicle-treated K562 cells were treated with 10 μM of C 17 sphingosine for 20 minutes. Then, we replaced the supernatant with PBS containing 0.5% BSA and incubated the cells at 37°C for another 20 minutes. Then, the supernatants and cells were collected and used for the C 17 S1P measurements (n = 6/group). (E) The expression of key enzymes in S1P metabolism was determined using real-time PCR. GAPDH was utilized as an internal control (n = 8/group). (F) SK activity assay. The SK activity assay was performed using NaB-treated or vehicle-treated K562 cells (n = 6/group). (G) Reverse transcription PCR was performed using cDNAs prepared from NaB-treated K562 cells (K), HepG2 cells (He), and HUVECs (Hu). (H) The expression of possible S1P transporters was determined using real-time PCR. GAPDH was utilized as an internal control (n = 4/group). (I) Real-time PCR of Band3. GAPDH was utilized as an internal control (n = 8/group). (J) Western blot of Band3 with membranous protein. The whole cell lysate of RBCs (2 μg) was placed as a positive control. Pan-cadherin was utilized as an internal control (n = 3/group).

    Journal: PLoS ONE

    Article Title: Involvement of Band3 in the efflux of sphingosine 1-phosphate from erythrocytes

    doi: 10.1371/journal.pone.0177543

    Figure Lengend Snippet: Modulation of S1P homeostasis of K562 cells by sodium butyrate-induced differentiation into erythroblast-like cells. K562 cells were treated with 2 mM sodium butyrate (NaB); 72 hours later, we investigated the modulation of S1P homeostasis. (A) The mRNA levels of GYPA and ALAS were determined using real-time PCR. GAPDH was utilized as an internal control (n = 8/group). (B, C) The expression of CD235a was investigated with a flow cytometer (n = 3/group). (D) C 17 S1P formation assay. NaB-treated or vehicle-treated K562 cells were treated with 10 μM of C 17 sphingosine for 20 minutes. Then, we replaced the supernatant with PBS containing 0.5% BSA and incubated the cells at 37°C for another 20 minutes. Then, the supernatants and cells were collected and used for the C 17 S1P measurements (n = 6/group). (E) The expression of key enzymes in S1P metabolism was determined using real-time PCR. GAPDH was utilized as an internal control (n = 8/group). (F) SK activity assay. The SK activity assay was performed using NaB-treated or vehicle-treated K562 cells (n = 6/group). (G) Reverse transcription PCR was performed using cDNAs prepared from NaB-treated K562 cells (K), HepG2 cells (He), and HUVECs (Hu). (H) The expression of possible S1P transporters was determined using real-time PCR. GAPDH was utilized as an internal control (n = 4/group). (I) Real-time PCR of Band3. GAPDH was utilized as an internal control (n = 8/group). (J) Western blot of Band3 with membranous protein. The whole cell lysate of RBCs (2 μg) was placed as a positive control. Pan-cadherin was utilized as an internal control (n = 3/group).

    Article Snippet: Then, we replaced the supernatant with PBS containing 0.5% fatty acid-free BSA (A8806; Sigma-Aldrich Co.) and incubated the cells at 37°C for another 20 minutes.

    Techniques: Real-time Polymerase Chain Reaction, Expressing, Flow Cytometry, Cytometry, Tube Formation Assay, Incubation, Activity Assay, Polymerase Chain Reaction, Western Blot, Positive Control

    Localisation of the hBD2 peptide in epithelial bronchial 16HBE cells . 16HBE cells were seeded at 5 × 10 5 cells per well in 1 ml of DMEM/F12 on 18-mm-diameter cover slips in 12 well plates in triplicate and grown for 16 h at 37°C. After washing the cover slips with PBS-BSA, the cells were exposed to either latex beads, ethanol fixed conidia or ethanol fixed HF for 18 hours. Il-1β was used as a positive control. Following washing with PBS, the cells were fixed with a paraformaldehyde solution for 30 min at 37°C. The slides were then incubated in 1% BSA/PBS-Triton 0.05%, followed by a solution of 10% normal goat serum. After washing, polyclonal rabbit anti-human hBD2 at a dilution of 1:250 was applied as primary antibody overnight at 4°C, followed by incubation with FITC-labelled goat anti-rabbit secondary antibody at a dilution of 1:300 for 4 hours at room temperature. After washing, the cover slips were mounted on slides with ProLong antifade Vectashield. Samples were viewed with a Zeiss fluorescence microscope using ×400 magnification. The arrows indicate the cells stained with anti-hBD2 antibody. The percentage of stained cells was computed from triplicates of four experiments. Means followed by the same letter are not significantly different. +, presence; -, absence of Il-1β, A. fumigatus fixed organisms and latex beads. The punctuated localisation of the signal, which is concentrated adjacent to the nucleus (arrow), was observed. The data shown are representative of four independent experiments.

    Journal: BMC Microbiology

    Article Title: Inducible expression of beta defensins by human respiratory epithelial cells exposed to Aspergillus fumigatus organisms

    doi: 10.1186/1471-2180-9-33

    Figure Lengend Snippet: Localisation of the hBD2 peptide in epithelial bronchial 16HBE cells . 16HBE cells were seeded at 5 × 10 5 cells per well in 1 ml of DMEM/F12 on 18-mm-diameter cover slips in 12 well plates in triplicate and grown for 16 h at 37°C. After washing the cover slips with PBS-BSA, the cells were exposed to either latex beads, ethanol fixed conidia or ethanol fixed HF for 18 hours. Il-1β was used as a positive control. Following washing with PBS, the cells were fixed with a paraformaldehyde solution for 30 min at 37°C. The slides were then incubated in 1% BSA/PBS-Triton 0.05%, followed by a solution of 10% normal goat serum. After washing, polyclonal rabbit anti-human hBD2 at a dilution of 1:250 was applied as primary antibody overnight at 4°C, followed by incubation with FITC-labelled goat anti-rabbit secondary antibody at a dilution of 1:300 for 4 hours at room temperature. After washing, the cover slips were mounted on slides with ProLong antifade Vectashield. Samples were viewed with a Zeiss fluorescence microscope using ×400 magnification. The arrows indicate the cells stained with anti-hBD2 antibody. The percentage of stained cells was computed from triplicates of four experiments. Means followed by the same letter are not significantly different. +, presence; -, absence of Il-1β, A. fumigatus fixed organisms and latex beads. The punctuated localisation of the signal, which is concentrated adjacent to the nucleus (arrow), was observed. The data shown are representative of four independent experiments.

    Article Snippet: After washing the cover slips with 5% BSA/PBS (BSA, Fraction V, Sigma), the cells were exposed to either 106 fixed conidia or to 20 μl of the fixed HF solution (20 mg of dry weight/ml), or 5 × 106 latex beads for 24 hours.

    Techniques: Positive Control, Incubation, Fluorescence, Microscopy, Staining

    Analysis of the defensin expression and its localisation in pneumocytes A549 exposed to live A. fumigatus . A . RT-PCR analysis of defensin mRNA expression by human pneumocyte A549 cells exposed to live A. fumigatus . A549 human epithelial bronchial cells (5 × 10 6 ) were grown in six well plates for 24 hours. The cells were then exposed either to live A. fumigatus conidia or latex beads. After 18 hours of incubation, the cells were washed with PBS, mRNA was isolated by TRIzol Reagent, and RT-PCR was performed as described above in Methods. Specific primer pairs (Table 1) were used for RNA amplification. The size of the amplified product is indicated and was as predicted. Cells were cultivated in a control well in the absence of A. fumigatus . As an additional control, the cells were exposed to 10 6 latex beads for the same period. GAPDH was uniformly expressed. One of the four results is shown. B . Immunofluorescence detection of hBD2 in the A549 exposed to live A. fumigatus conidia. A549 cells were seeded at 5 × 10 5 cells per well in 1 ml of DMEM/F12 on 18-mm-diameter cover slips in 12 well plates in triplicate and grown for 16 h at 37°C. After washing the cover slips with 1%BSA/PBS, the cells were exposed to either latex beads or live A. fumigatus conidia for 18 hours. Il-1β was used as a positive control. Some cells were treated with TNF-α. Following washing with PBS, the cells were fixed with a paraformaldehyde solution for 30 min at 37°C. The slides were then incubated in 1% BSA/PBS, followed by a solution of 10% normal goat serum. After washing, polyclonal rabbit anti-human hBD2 at a dilution of 1:250 was applied as primary antibody overnight at 4°C, followed by incubation with FITC-labelled goat anti-rabbit secondary antibody at a dilution of 1:300 for 4 hours at room temperature. After washing, the cover slips were mounted on slides with ProLong antifade Vectashield. Samples were viewed with a Zeiss fluorescence microscope using ×400 magnification. The arrows indicate the cells stained with anti-hBD2 antibody. The percentage of stained cells was computed from triplicates of four experiments. Means followed by the same letter are not significantly different. +, presence; -, absence of Il-1β, TNF-α, live A. fumigatus organism and latex beads. The punctuated localisation of the signal, which is concentrated adjacent to the nucleus (arrow), was observed. The data shown are representative of four independent experiments.

    Journal: BMC Microbiology

    Article Title: Inducible expression of beta defensins by human respiratory epithelial cells exposed to Aspergillus fumigatus organisms

    doi: 10.1186/1471-2180-9-33

    Figure Lengend Snippet: Analysis of the defensin expression and its localisation in pneumocytes A549 exposed to live A. fumigatus . A . RT-PCR analysis of defensin mRNA expression by human pneumocyte A549 cells exposed to live A. fumigatus . A549 human epithelial bronchial cells (5 × 10 6 ) were grown in six well plates for 24 hours. The cells were then exposed either to live A. fumigatus conidia or latex beads. After 18 hours of incubation, the cells were washed with PBS, mRNA was isolated by TRIzol Reagent, and RT-PCR was performed as described above in Methods. Specific primer pairs (Table 1) were used for RNA amplification. The size of the amplified product is indicated and was as predicted. Cells were cultivated in a control well in the absence of A. fumigatus . As an additional control, the cells were exposed to 10 6 latex beads for the same period. GAPDH was uniformly expressed. One of the four results is shown. B . Immunofluorescence detection of hBD2 in the A549 exposed to live A. fumigatus conidia. A549 cells were seeded at 5 × 10 5 cells per well in 1 ml of DMEM/F12 on 18-mm-diameter cover slips in 12 well plates in triplicate and grown for 16 h at 37°C. After washing the cover slips with 1%BSA/PBS, the cells were exposed to either latex beads or live A. fumigatus conidia for 18 hours. Il-1β was used as a positive control. Some cells were treated with TNF-α. Following washing with PBS, the cells were fixed with a paraformaldehyde solution for 30 min at 37°C. The slides were then incubated in 1% BSA/PBS, followed by a solution of 10% normal goat serum. After washing, polyclonal rabbit anti-human hBD2 at a dilution of 1:250 was applied as primary antibody overnight at 4°C, followed by incubation with FITC-labelled goat anti-rabbit secondary antibody at a dilution of 1:300 for 4 hours at room temperature. After washing, the cover slips were mounted on slides with ProLong antifade Vectashield. Samples were viewed with a Zeiss fluorescence microscope using ×400 magnification. The arrows indicate the cells stained with anti-hBD2 antibody. The percentage of stained cells was computed from triplicates of four experiments. Means followed by the same letter are not significantly different. +, presence; -, absence of Il-1β, TNF-α, live A. fumigatus organism and latex beads. The punctuated localisation of the signal, which is concentrated adjacent to the nucleus (arrow), was observed. The data shown are representative of four independent experiments.

    Article Snippet: After washing the cover slips with 5% BSA/PBS (BSA, Fraction V, Sigma), the cells were exposed to either 106 fixed conidia or to 20 μl of the fixed HF solution (20 mg of dry weight/ml), or 5 × 106 latex beads for 24 hours.

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Incubation, Isolation, Amplification, Immunofluorescence, Positive Control, Fluorescence, Microscopy, Staining

    Effects of hyaluronidase on the ability of supernatants from ECs to activate neutrophils morphologically. ECs were cultured for 3 or 20 days and treated with 1 or 100 U/ml TNF for 4 hours. Medium was removed and replaced with PBS/BSA with or without 30 mU/ml hyaluronidase for 30 minutes. The supernatants were collected and added to neutrophils for 5 minutes and their shape change was observed using a microscope to determine the percent activated. For comparison, neutrophils were also treated with PBS/BSA with or without hyaluronidase. Data are mean ± SEM for 3 experiments. ⁎p

    Journal: Experimental Cell Research

    Article Title: A role for the endothelial glycosaminoglycan hyaluronan in neutrophil recruitment by endothelial cells cultured for prolonged periods

    doi: 10.1016/j.yexcr.2009.08.012

    Figure Lengend Snippet: Effects of hyaluronidase on the ability of supernatants from ECs to activate neutrophils morphologically. ECs were cultured for 3 or 20 days and treated with 1 or 100 U/ml TNF for 4 hours. Medium was removed and replaced with PBS/BSA with or without 30 mU/ml hyaluronidase for 30 minutes. The supernatants were collected and added to neutrophils for 5 minutes and their shape change was observed using a microscope to determine the percent activated. For comparison, neutrophils were also treated with PBS/BSA with or without hyaluronidase. Data are mean ± SEM for 3 experiments. ⁎p

    Article Snippet: The monolayer was then treated with 2 ml of PBS/BSA with or without 30 mU/ml heparinase (Oxford Glycosciences, Oxford, UK) or 30 U/ml hyaluronidase (Sigma) for 15 minutes immediately prior to the assay.

    Techniques: Cell Culture, Microscopy

    Effect of heparinase treatment of ECs on (A) adhesion or (B) transmigration of neutrophils. ECs were cultured for 20 days, stimulated with 1 or 100 U/ml TNF for 4 hours, then treated with PBS/BSA with or without 30 mU/ml heparinase for 30 minutes prior to assay. Adhesion and transmigration efficiency are shown for heparinase-treated ECs relative to untreated ECs. Data are mean ± SEM from 3 experiments.

    Journal: Experimental Cell Research

    Article Title: A role for the endothelial glycosaminoglycan hyaluronan in neutrophil recruitment by endothelial cells cultured for prolonged periods

    doi: 10.1016/j.yexcr.2009.08.012

    Figure Lengend Snippet: Effect of heparinase treatment of ECs on (A) adhesion or (B) transmigration of neutrophils. ECs were cultured for 20 days, stimulated with 1 or 100 U/ml TNF for 4 hours, then treated with PBS/BSA with or without 30 mU/ml heparinase for 30 minutes prior to assay. Adhesion and transmigration efficiency are shown for heparinase-treated ECs relative to untreated ECs. Data are mean ± SEM from 3 experiments.

    Article Snippet: The monolayer was then treated with 2 ml of PBS/BSA with or without 30 mU/ml heparinase (Oxford Glycosciences, Oxford, UK) or 30 U/ml hyaluronidase (Sigma) for 15 minutes immediately prior to the assay.

    Techniques: Transmigration Assay, Cell Culture

    Effect of hyaluronidase treatment of ECs on (A) adhesion or (B) transmigration of neutrophils. ECs were cultured for 3 or 20 days, stimulated with 1 or 100 U/ml TNF for 4 hours, then treated with PBS/BSA with or without 30 mU/ml hyaluronidase for 30 minutes prior to the assay. Adhesion and transmigration efficiency are shown for heparinase-treated ECs relative to untreated ECs. Data are mean ± SEM from 4 experiments. ⁎⁎p

    Journal: Experimental Cell Research

    Article Title: A role for the endothelial glycosaminoglycan hyaluronan in neutrophil recruitment by endothelial cells cultured for prolonged periods

    doi: 10.1016/j.yexcr.2009.08.012

    Figure Lengend Snippet: Effect of hyaluronidase treatment of ECs on (A) adhesion or (B) transmigration of neutrophils. ECs were cultured for 3 or 20 days, stimulated with 1 or 100 U/ml TNF for 4 hours, then treated with PBS/BSA with or without 30 mU/ml hyaluronidase for 30 minutes prior to the assay. Adhesion and transmigration efficiency are shown for heparinase-treated ECs relative to untreated ECs. Data are mean ± SEM from 4 experiments. ⁎⁎p

    Article Snippet: The monolayer was then treated with 2 ml of PBS/BSA with or without 30 mU/ml heparinase (Oxford Glycosciences, Oxford, UK) or 30 U/ml hyaluronidase (Sigma) for 15 minutes immediately prior to the assay.

    Techniques: Transmigration Assay, Cell Culture

    Valsartan or aliskiren prevented palmitic acid (PA; 0.8 mM)-induced endoplasmic reticulum (ER) stress in cultured human proximal tubule epithelial cells (HK2) cells after a 24-h treatment. A and B : protein abundance of binding immunoglobulin protein (BiP) and C/EBP homologous protein (CHOP) was unchanged in HK2 cells treated with BSA (2 and 10 mg/ml) and with or without valsartan (10 −6 M) or aliskiren (10 −7 M). C : MTT assays of HK2 cells. HK2 cells were incubated with PA at different concentrations for 24 h. After the cells were incubated with tetrazolium salt solution for 2 h, the quantity of formazan product was determined from the absorbance at 560 nm. * P

    Journal: American Journal of Physiology - Renal Physiology

    Article Title: Intrarenal renin-angiotensin system mediates fatty acid-induced ER stress in the kidney

    doi: 10.1152/ajprenal.00223.2015

    Figure Lengend Snippet: Valsartan or aliskiren prevented palmitic acid (PA; 0.8 mM)-induced endoplasmic reticulum (ER) stress in cultured human proximal tubule epithelial cells (HK2) cells after a 24-h treatment. A and B : protein abundance of binding immunoglobulin protein (BiP) and C/EBP homologous protein (CHOP) was unchanged in HK2 cells treated with BSA (2 and 10 mg/ml) and with or without valsartan (10 −6 M) or aliskiren (10 −7 M). C : MTT assays of HK2 cells. HK2 cells were incubated with PA at different concentrations for 24 h. After the cells were incubated with tetrazolium salt solution for 2 h, the quantity of formazan product was determined from the absorbance at 560 nm. * P

    Article Snippet: PA, fatty acid-free bovine serum albumin (BSA), tunicamycin, and MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-dipthenyltetrazolium bromide) were purchased from Sigma-Aldrich; anti-BiP (3177), anti-CHOP (2895), anti-peIF2α (3597)/eIF2α (9722), anti-IRE1α (3294), and anti-activated caspase-3 (9664) antibodies from Cell Signaling; anti-GRP78 (BiP) (SC-13968 for immunohistochemistry) from Santa-Cruz Biotechnology; anti-ATF4 from Abcam (ab50546); and anti-β-actin from Sigma.

    Techniques: Cell Culture, Binding Assay, MTT Assay, Incubation

    Effects of BSA and HSA on cytokine release from human adipose tissue. Adipose tissue was incubated for 2, 4, 6, 8, and 24 h in albumin-free medium (blue) and medium supplemented with 1% HSA (red), 1% BSA Fraction V (purple), or 1% BSA Essentially Fatty Acid Free (black). Concentrations of nine cytokines in the conditioned media were analyzed with a multiplex immunoassay; (A) granulocyte-macrophage colony-stimulating factor (B) interferon-γ (C) IL-1β (D) IL-2 (E) IL-6 (F) IL-8 (G) IL-10 (H) IL-12p70 (I) TNF-α. Insets show detailed plots of cytokine release in medium without albumin and in medium with HSA. Concentrations are ng/mL/g adipose tissue for IL-6 and IL-8 and pg/mL/g adipose tissue for the remaining cytokines. Values mean ± SEM (n = 3). Both media containing BSA markedly induced the release of all nine cytokines vs albumin-free medium or medium containing HSA over 24 h (p

    Journal: BMC Endocrine Disorders

    Article Title: Adiponectin, chemerin, cytokines, and dipeptidyl peptidase 4 are released from human adipose tissue in a depot-dependent manner: an in vitro system including human serum albumin

    doi: 10.1186/1472-6823-14-7

    Figure Lengend Snippet: Effects of BSA and HSA on cytokine release from human adipose tissue. Adipose tissue was incubated for 2, 4, 6, 8, and 24 h in albumin-free medium (blue) and medium supplemented with 1% HSA (red), 1% BSA Fraction V (purple), or 1% BSA Essentially Fatty Acid Free (black). Concentrations of nine cytokines in the conditioned media were analyzed with a multiplex immunoassay; (A) granulocyte-macrophage colony-stimulating factor (B) interferon-γ (C) IL-1β (D) IL-2 (E) IL-6 (F) IL-8 (G) IL-10 (H) IL-12p70 (I) TNF-α. Insets show detailed plots of cytokine release in medium without albumin and in medium with HSA. Concentrations are ng/mL/g adipose tissue for IL-6 and IL-8 and pg/mL/g adipose tissue for the remaining cytokines. Values mean ± SEM (n = 3). Both media containing BSA markedly induced the release of all nine cytokines vs albumin-free medium or medium containing HSA over 24 h (p

    Article Snippet: To evaluate the influence of albumin preparation, exploratory incubations for 2, 4, 6, 8, and 24 h were carried out in medium lacking albumin and in medium supplemented with 1% HSA, 1% BSA Fraction V, or 1% BSA Essentially Fatty Acid Free (Sigma-Aldrich, St Louis, MO).

    Techniques: Incubation, Multiplex Assay

    DC-derived IL-15 controls granuloma formation. (A) Spleen cells were obtained from DTR tg mice 24 h after injection of 100 ng DT or PBS and stained with anti-CD11c–PE. The percentages indicate the proportions of DCs in a DC-enriched fraction; i.e., the cells at the interface after a dense BSA gradient centrifugation (see DC preparation… in Materials and methods). (B) Granuloma formation in the liver of P. acnes –primed DT-injected DTR tg mice. On day 3 after a 0.5-mg heat-killed P. acnes injection, livers were taken from WT and DTR tg mice that had been injected with either PBS or DT on the day before P. acnes injection, and sections were stained with H E. Numbers of granulomas were counted in five different fields under a microscope. Values represent SD ( n = 3 mice/group). Data are representative of three experiments. (C) To test whether DCs were present in the granuloma regions, the liver sections were stained with biotinylated anti-CD11c mAb and streptavidin-HRP and further visualized with DAB. Slides were counterstained with Mayer's hematoxylin. Bar, 100 μm. (D) IL-12p70, IFN-γ, and chemokine levels in the sera of DT-injected DTR tg mice were assessed by ELISA at 72 h after a 0.5-mg heat-killed P. acnes injection. Values represent SD ( n = 3 mice/group). Data are representative of three experiments. (E) Immunofluoresence staining for the identification of IL-15–producing cells. Acetone-fixed frozen tissue sections were incubated with FITC-conjugated anti-CD11c (clone N418) and biotinylated anti–IL-15 antibodies and further developed with streptavidin-PE. Bar, 50 μm. (F) Granuloma formation in the liver of zymosan-primed DT-injected DTR tg mice. As described in B and C, livers were taken from the indicated mice on day 3 after a 1-mg zymosan injection, and sections were stained for CD11c. Slides were counterstained with Mayer's hematoxylin. Bar, 100 μm. (G) Granuloma formation in the liver of BMDC-injected IL-15 −/− mice. IL-15 −/− mice were injected with 1 × 10 6 WT BMDCs or IL-15 −/− BMDCs. After 12 h, mice were injected with 0.5 mg P. acnes , and granuloma formation was analyzed 6 d later as described in B, C, and F. Bar, 100 μm.

    Journal: The Journal of Experimental Medicine

    Article Title: Essential roles of DC-derived IL-15 as a mediator of inflammatory responses in vivo

    doi: 10.1084/jem.20061297

    Figure Lengend Snippet: DC-derived IL-15 controls granuloma formation. (A) Spleen cells were obtained from DTR tg mice 24 h after injection of 100 ng DT or PBS and stained with anti-CD11c–PE. The percentages indicate the proportions of DCs in a DC-enriched fraction; i.e., the cells at the interface after a dense BSA gradient centrifugation (see DC preparation… in Materials and methods). (B) Granuloma formation in the liver of P. acnes –primed DT-injected DTR tg mice. On day 3 after a 0.5-mg heat-killed P. acnes injection, livers were taken from WT and DTR tg mice that had been injected with either PBS or DT on the day before P. acnes injection, and sections were stained with H E. Numbers of granulomas were counted in five different fields under a microscope. Values represent SD ( n = 3 mice/group). Data are representative of three experiments. (C) To test whether DCs were present in the granuloma regions, the liver sections were stained with biotinylated anti-CD11c mAb and streptavidin-HRP and further visualized with DAB. Slides were counterstained with Mayer's hematoxylin. Bar, 100 μm. (D) IL-12p70, IFN-γ, and chemokine levels in the sera of DT-injected DTR tg mice were assessed by ELISA at 72 h after a 0.5-mg heat-killed P. acnes injection. Values represent SD ( n = 3 mice/group). Data are representative of three experiments. (E) Immunofluoresence staining for the identification of IL-15–producing cells. Acetone-fixed frozen tissue sections were incubated with FITC-conjugated anti-CD11c (clone N418) and biotinylated anti–IL-15 antibodies and further developed with streptavidin-PE. Bar, 50 μm. (F) Granuloma formation in the liver of zymosan-primed DT-injected DTR tg mice. As described in B and C, livers were taken from the indicated mice on day 3 after a 1-mg zymosan injection, and sections were stained for CD11c. Slides were counterstained with Mayer's hematoxylin. Bar, 100 μm. (G) Granuloma formation in the liver of BMDC-injected IL-15 −/− mice. IL-15 −/− mice were injected with 1 × 10 6 WT BMDCs or IL-15 −/− BMDCs. After 12 h, mice were injected with 0.5 mg P. acnes , and granuloma formation was analyzed 6 d later as described in B, C, and F. Bar, 100 μm.

    Article Snippet: In brief, collagenase-digested spleen cells were suspended in a 28% BSA solution in 1.08 g/ml PBS, overlaid with 1 ml FCS-free RPMI 1640 medium (Sigma-Aldrich), and centrifuged at 9,500 g for 20 min at 4°C.

    Techniques: Derivative Assay, Mouse Assay, Injection, Staining, Gradient Centrifugation, Microscopy, Enzyme-linked Immunosorbent Assay, Incubation

    Selectivity evaluation of the biosensor for the detection of thrombin against other proteins of Lysozyme, Trypsase, lgG, BSA, and HSA. The concentration of the thrombin was 0.2 µM. However, the concentration of Lysozyme, Trypsase, lgG, BSA, and HSA was 2 µM. No thrombin or other proteins were used in the blank group. Inset: the changes in fluorescence intensity (F/F 0 ), F and F 0 , are the fluorescence intensities in the presence and absence of protein, respectively. The error bar was calculated in three independent experiments.

    Journal: Sensors (Basel, Switzerland)

    Article Title: An Enzyme- and Label-Free Fluorescence Aptasensor for Detection of Thrombin Based on Graphene Oxide and G-Quadruplex

    doi: 10.3390/s19204424

    Figure Lengend Snippet: Selectivity evaluation of the biosensor for the detection of thrombin against other proteins of Lysozyme, Trypsase, lgG, BSA, and HSA. The concentration of the thrombin was 0.2 µM. However, the concentration of Lysozyme, Trypsase, lgG, BSA, and HSA was 2 µM. No thrombin or other proteins were used in the blank group. Inset: the changes in fluorescence intensity (F/F 0 ), F and F 0 , are the fluorescence intensities in the presence and absence of protein, respectively. The error bar was calculated in three independent experiments.

    Article Snippet: Lysozyme, thrombin, lgG, human serum albumin (HSA), and bovine serum albumin (BSA) were purchased from Sigma (St. Louis, MO, USA).

    Techniques: Concentration Assay, Fluorescence

    The ARC CARD is required for the ability of ARC to suppress TNF α -induced necrosis. ( a ) Immunoblot showing inhibition of TNF α -induced HMGB1 release by wild-type ARC but not by the CARD-defective double-point ARC mutant (L31F; G69R). L929 cells were stably transduced with empty vector (Φ), ARC-HA (ARC) or ARC-HA double-point CARD mutant (DM). Immunblot of bovine serum albumin (BSA) in media used as loading control. ( b ) Inhibition of TNF α -induced LDH release by ARC requires the CARD. Data shown as mean±S.E. from n =4. *** P -value

    Journal: Cell Death and Differentiation

    Article Title: A novel role for the apoptosis inhibitor ARC in suppressing TNFα-induced regulated necrosis

    doi: 10.1038/cdd.2013.195

    Figure Lengend Snippet: The ARC CARD is required for the ability of ARC to suppress TNF α -induced necrosis. ( a ) Immunoblot showing inhibition of TNF α -induced HMGB1 release by wild-type ARC but not by the CARD-defective double-point ARC mutant (L31F; G69R). L929 cells were stably transduced with empty vector (Φ), ARC-HA (ARC) or ARC-HA double-point CARD mutant (DM). Immunblot of bovine serum albumin (BSA) in media used as loading control. ( b ) Inhibition of TNF α -induced LDH release by ARC requires the CARD. Data shown as mean±S.E. from n =4. *** P -value

    Article Snippet: Primary antibodies include: ARC (Cayman Chemical, Ann Arbor, MI, USA); β -actin (Sigma-Aldrich, St. Louis, MO, USA); HA (Roche Applied Science, Indianapolis, IN, USA); BSA, HMGB1, His (Abcam, Cambridge, MA, USA); histone H3, PARP, p65 (Cell Signaling Technology, Danvers, MA, USA); RhoGDI α (A-20) (Santa Cruz Biotechnology, Dallas, TX, USA); mouse RIP3 (Prosci, Poway, CA, USA); human RIP3 (Thermo Scientific); RIP1 (BD Biosciences, San Jose, CA, USA); and FADD (Enzo Life Sciences, Farmingdale, NY, USA).

    Techniques: Inhibition, Mutagenesis, Stable Transfection, Transduction, Plasmid Preparation

    GDF11-induced phenotypes require SMAD4 and ID2 (A) SMAD4 knockdown blocks the ability of GDF11 to inhibit 3D invasion in MDA-MB-231 cells. shLuc was induced as a control shRNA. (B) Inducible reconstitution of SMAD4 in SMAD4-null MDA-MB-468 basal-like TNBC cells. FLAG-LacZ was induced as a control overexpression. MCF10A-5E cell extracts were included to gauge endogenous abundance of SMAD4. (C) SMAD4 reconstitution is sufficient to confer GDF11 responsiveness to MDA-MB-468 cells. Control cells express inducible FLAG-LacZ. (D) Differential transcriptomic profiling of GDF11- and TGFβ-stimulated NMuMG 3D cultures reveals Id2 as a candidate GDF11 effector. NMuMG spheroids were grown for 6 days in culture and stimulated with either cytokine for 4 hours and RNA expression was measured by Illumina BeadChip arrays. Representative data are shown from n = 4 biological replicates. (E) Knockdown of Id2 blocks the ability of GDF11 to promote 3D rounding in NMuMG cells. shGFP was induced as a control shRNA. (F) ID2 knockdown blocks the ability of GDF11 to inhibit 3D invasion in MDA-MB-231 cells. shLuc was induced as a control shRNA. (G) Basal-like breast cancer (BLBC) patients with GDF11 expression ( > 1.5 copies per cell) and elevated ACVR2B and TGFBR3 show increased abundance of ID2 ). (H) Tumor bioluminescence of MDA-MB-231 intraductal xenografts co-inoculated with 200 ng GDF11 or BSA control. Tumor bioluminescence was measured at day 2, and every seven days post inoculation. (I and J) Recombinant GDF11 reduces intraductal proliferation but does not affect apoptosis. Intraductal lesions were stained for Ki67 (I) or cleaved caspase-3 (ClvC3), and positive cells per 10× field of view (FOV) were quantified for paired GDF11 and control glands (J). (K) ID2 knockdown blocks the ability of GDF11 to suppress intraductal growth of MDA-MB-231 cells. shLacZ was induced as a control shRNA with shID2 #2 at day 10. Data are shown as the mean ± SEM of n = 4 (A, C, E, F) biological replicates or 10 mice (K) or the median ± 90% nonparametric confidence interval of n ). 3D cultures were assessed at day 16 (A, F), 12 (C), and 20 (E). Scale bar is 200 μm (A, C, F) and 40 μm (I). n.s., not significant ( p > 0.05 by two-sided Ward’s test [A, F] or sign-rank test [J]). .

    Journal: Developmental cell

    Article Title: Tumor Suppressor Inactivation of GDF11 Occurs by Precursor Sequestration in Triple-Negative Breast Cancer

    doi: 10.1016/j.devcel.2017.10.027

    Figure Lengend Snippet: GDF11-induced phenotypes require SMAD4 and ID2 (A) SMAD4 knockdown blocks the ability of GDF11 to inhibit 3D invasion in MDA-MB-231 cells. shLuc was induced as a control shRNA. (B) Inducible reconstitution of SMAD4 in SMAD4-null MDA-MB-468 basal-like TNBC cells. FLAG-LacZ was induced as a control overexpression. MCF10A-5E cell extracts were included to gauge endogenous abundance of SMAD4. (C) SMAD4 reconstitution is sufficient to confer GDF11 responsiveness to MDA-MB-468 cells. Control cells express inducible FLAG-LacZ. (D) Differential transcriptomic profiling of GDF11- and TGFβ-stimulated NMuMG 3D cultures reveals Id2 as a candidate GDF11 effector. NMuMG spheroids were grown for 6 days in culture and stimulated with either cytokine for 4 hours and RNA expression was measured by Illumina BeadChip arrays. Representative data are shown from n = 4 biological replicates. (E) Knockdown of Id2 blocks the ability of GDF11 to promote 3D rounding in NMuMG cells. shGFP was induced as a control shRNA. (F) ID2 knockdown blocks the ability of GDF11 to inhibit 3D invasion in MDA-MB-231 cells. shLuc was induced as a control shRNA. (G) Basal-like breast cancer (BLBC) patients with GDF11 expression ( > 1.5 copies per cell) and elevated ACVR2B and TGFBR3 show increased abundance of ID2 ). (H) Tumor bioluminescence of MDA-MB-231 intraductal xenografts co-inoculated with 200 ng GDF11 or BSA control. Tumor bioluminescence was measured at day 2, and every seven days post inoculation. (I and J) Recombinant GDF11 reduces intraductal proliferation but does not affect apoptosis. Intraductal lesions were stained for Ki67 (I) or cleaved caspase-3 (ClvC3), and positive cells per 10× field of view (FOV) were quantified for paired GDF11 and control glands (J). (K) ID2 knockdown blocks the ability of GDF11 to suppress intraductal growth of MDA-MB-231 cells. shLacZ was induced as a control shRNA with shID2 #2 at day 10. Data are shown as the mean ± SEM of n = 4 (A, C, E, F) biological replicates or 10 mice (K) or the median ± 90% nonparametric confidence interval of n ). 3D cultures were assessed at day 16 (A, F), 12 (C), and 20 (E). Scale bar is 200 μm (A, C, F) and 40 μm (I). n.s., not significant ( p > 0.05 by two-sided Ward’s test [A, F] or sign-rank test [J]). .

    Article Snippet: Membranes were blocked with 1% (w/v) BSA in TBS-T for one hour at room temperature and then incubated overnight at 4°C with GDF11–EPR antibody (EPR4567(2), Abcam #ab124721, 1:1000) or GDF11–1E6 antibody (1E6, Novus Biological #H00010220-M03, 1:500) diluted in 1% (w/v) BSA in TBS-T. Membranes were washed 3 × 5 minutes in TBS-T and then incubated for one hour at room temperature with HRP-conjugated goat anti-rabbit or anti-mouse secondary antibody (Jackson ImmunoResearch #111-035-144 or #115-035-146, 1:10,000) diluted in 1% (w/v) BSA in TBS-T. Membranes were again washed 3 × 5 minutes in TBS-T and exposed by chemiluminescence with SuperSignal West Femto (Thermo Scientific) as described above.

    Techniques: Multiple Displacement Amplification, shRNA, Over Expression, RNA Expression, Expressing, Recombinant, Staining, Mouse Assay

    Purification of anti-BSA-digoxin bispecific antibodies. Anti-BSA-digoxin bispecific antibodies were purified from the serum using a series of purification methods. IgG fraction was obtained using 50% saturated ammonium sulfate precipitation, followed by passing through a protein A column. Bispecific antibody was further purified by affinity chromatography, using digoxin-Sepharose and BSA-Sepharose, after which a pure antibody fraction possessing bispecificity was obtained. Monospecific and bispecific antibody ELISAs were performed to monitor antibody titers during the entire purification process and the results were plotted.

    Journal: PLoS ONE

    Article Title: Production of Native Bispecific Antibodies in Rabbits

    doi: 10.1371/journal.pone.0010879

    Figure Lengend Snippet: Purification of anti-BSA-digoxin bispecific antibodies. Anti-BSA-digoxin bispecific antibodies were purified from the serum using a series of purification methods. IgG fraction was obtained using 50% saturated ammonium sulfate precipitation, followed by passing through a protein A column. Bispecific antibody was further purified by affinity chromatography, using digoxin-Sepharose and BSA-Sepharose, after which a pure antibody fraction possessing bispecificity was obtained. Monospecific and bispecific antibody ELISAs were performed to monitor antibody titers during the entire purification process and the results were plotted.

    Article Snippet: Conjugation of BSA to CNBr-activated Sepharose 4B (GE Healthcare, Amersham Biosciences, Sweden) was conducted according to the manufacturer's instructions.

    Techniques: Purification, Affinity Chromatography

    PCR amplification with Neq DNA polymerase. (a) Effect of pH on the PCR amplification with Neq DNA polymerase. The amplification of the 1-kb λ DNA fragment was performed in a 50-μl reaction mixture containing 50 mM Tris-HCl, 2 mM MgCl 2 , 50 mM KCl, and 0.01% BSA at the indicated pH values. (b) Effect of MgCl 2 on the PCR amplification with Neq DNA polymerase. The amplification of the 1-kb λ DNA fragment was performed in a 50-μl reaction mixture containing 50 mM Tris-HCl (pH 8.0), 50 mM KCl, and 0.01% BSA with the indicated concentrations of MgCl 2 . (c) Effect of KCl on the PCR amplification with Neq DNA polymerase. The amplification of the 1-kb λ DNA fragment was performed in a 50-μl reaction mixture containing 50 mM Tris-HCl (pH 8.0), 2 mM MgCl 2 , and 0.01% BSA with the indicated concentrations of KCl. Lane M, DNA molecular size markers.

    Journal: Applied and Environmental Microbiology

    Article Title: Unique Substrate Spectrum and PCR Application of Nanoarchaeum equitans Family B DNA Polymerase ▿

    doi: 10.1128/AEM.00624-08

    Figure Lengend Snippet: PCR amplification with Neq DNA polymerase. (a) Effect of pH on the PCR amplification with Neq DNA polymerase. The amplification of the 1-kb λ DNA fragment was performed in a 50-μl reaction mixture containing 50 mM Tris-HCl, 2 mM MgCl 2 , 50 mM KCl, and 0.01% BSA at the indicated pH values. (b) Effect of MgCl 2 on the PCR amplification with Neq DNA polymerase. The amplification of the 1-kb λ DNA fragment was performed in a 50-μl reaction mixture containing 50 mM Tris-HCl (pH 8.0), 50 mM KCl, and 0.01% BSA with the indicated concentrations of MgCl 2 . (c) Effect of KCl on the PCR amplification with Neq DNA polymerase. The amplification of the 1-kb λ DNA fragment was performed in a 50-μl reaction mixture containing 50 mM Tris-HCl (pH 8.0), 2 mM MgCl 2 , and 0.01% BSA with the indicated concentrations of KCl. Lane M, DNA molecular size markers.

    Article Snippet: The 50-μl reaction mixture contained 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2 , 50 mM KCl, 0.01% bovine serum albumin (BSA), 200 μM each of dATP, dCTP, and dGTP, 20 μM dTTP, 0.5 μCi of [ methyl -3 H]TTP (30 Ci/mmol; GE Healthcare), 1.25 μg of activated calf thymus DNA, and enzyme solution.

    Techniques: Polymerase Chain Reaction, Amplification

    Cell polarity disrupts the cis interaction between P2Y 2 R and β 3 integrin and infectivity. (A) Confocal image of polarized Vero E6 cells expressing antibody-stained basolateral α v β 3 and apical P2Y 2 R. The top panel is a confocal image in which the focus plane was parallel to the monolayer ( XY scan), whereas the bottom panel shows the focus plane as a vertical cross section of the monolayer ( XZ scan). The white line indicated by the arrow in the XY scan indicates the path of the XZ scan. To enable visualization, Vero E6 cells were transiently transfected with P2Y 2 R and plated at confluence in eight-well Nunc Lab-Tek chambers and allowed to propagate for 1–2 d. At room temperature, cells were fixed with 3% paraformaldehyde for 20 min and subsequently permeabilized with 0.2% Triton X-100 (in PBS) for 15 min. Cells were blocked for nonspecific staining with 1% BSA in PBS for 30 min and then incubated with mouse monoclonal anti-integrin α v β 3 (1:40, Millipore MsxHu, MAB#1976) and rabbit polyclonal anti-P2Y 2 (1:100, Abcam, ab10270) were used with Alexa 488 and Alexa 580, respectively. (B) Plot of SNV FFU/ml vs. cells in suspension and on plates. CHO-A24, TIME, and Vero E6 cells were plated at confluence in vitronectin treated 48-well plates for 24 h. Confluent cells in triplicate wells were inoculated with 0.1 moi SNV for 1 h, washed in low pH media, and then incubated in normal media for 24 h. Media from infected cells was collected after 24 h diluted and subjected to a 7-day focus assay in Vero cells (see Supplemental Figure S1E for typical SNV FFU). ** p

    Journal: Molecular Biology of the Cell

    Article Title: Low-affinity binding in cis to P2Y2R mediates force-dependent integrin activation during hantavirus infection

    doi: 10.1091/mbc.E17-01-0082

    Figure Lengend Snippet: Cell polarity disrupts the cis interaction between P2Y 2 R and β 3 integrin and infectivity. (A) Confocal image of polarized Vero E6 cells expressing antibody-stained basolateral α v β 3 and apical P2Y 2 R. The top panel is a confocal image in which the focus plane was parallel to the monolayer ( XY scan), whereas the bottom panel shows the focus plane as a vertical cross section of the monolayer ( XZ scan). The white line indicated by the arrow in the XY scan indicates the path of the XZ scan. To enable visualization, Vero E6 cells were transiently transfected with P2Y 2 R and plated at confluence in eight-well Nunc Lab-Tek chambers and allowed to propagate for 1–2 d. At room temperature, cells were fixed with 3% paraformaldehyde for 20 min and subsequently permeabilized with 0.2% Triton X-100 (in PBS) for 15 min. Cells were blocked for nonspecific staining with 1% BSA in PBS for 30 min and then incubated with mouse monoclonal anti-integrin α v β 3 (1:40, Millipore MsxHu, MAB#1976) and rabbit polyclonal anti-P2Y 2 (1:100, Abcam, ab10270) were used with Alexa 488 and Alexa 580, respectively. (B) Plot of SNV FFU/ml vs. cells in suspension and on plates. CHO-A24, TIME, and Vero E6 cells were plated at confluence in vitronectin treated 48-well plates for 24 h. Confluent cells in triplicate wells were inoculated with 0.1 moi SNV for 1 h, washed in low pH media, and then incubated in normal media for 24 h. Media from infected cells was collected after 24 h diluted and subjected to a 7-day focus assay in Vero cells (see Supplemental Figure S1E for typical SNV FFU). ** p

    Article Snippet: Cells were blocked for nonspecific staining with 1% bovine serum albumin (BSA) in PBS for 30 min and then incubated with either anti-ZO1 (rabbit polyclonal from Abcam, 1:100 dilution in blocking buffer) or occludin (mouse mAb from Invitrogen, 1:200 dilution in blocking buffer) overnight at 4°C.

    Techniques: Infection, Expressing, Staining, Transfection, Incubation

    ). (D) ELISA sugar-binding assay for PdCoV S1-NTD. Here, the ELISA plates were precoated with sugar-rich mucin, and then PdCoV S1-NTD was added and incubated with mucin. Mucin-bound S1-NTD was detected using antibodies recognizing its C-terminal His 6 tag. Porcine epidemic diarrhea virus (PEDV) S1 was used as the positive control. PdCoV S1-CTD, SARS-CoV S1-CTD, and BSA were used as negative controls. A plate without mucin was used as an additional negative control. Statistical analyses were performed using two-tailed t test. Error bars indicate standard errors of the means (SEM) ( n = 5). ***, P

    Journal: Journal of Virology

    Article Title: Cryo-Electron Microscopy Structure of Porcine Deltacoronavirus Spike Protein in the Prefusion State

    doi: 10.1128/JVI.01556-17

    Figure Lengend Snippet: ). (D) ELISA sugar-binding assay for PdCoV S1-NTD. Here, the ELISA plates were precoated with sugar-rich mucin, and then PdCoV S1-NTD was added and incubated with mucin. Mucin-bound S1-NTD was detected using antibodies recognizing its C-terminal His 6 tag. Porcine epidemic diarrhea virus (PEDV) S1 was used as the positive control. PdCoV S1-CTD, SARS-CoV S1-CTD, and BSA were used as negative controls. A plate without mucin was used as an additional negative control. Statistical analyses were performed using two-tailed t test. Error bars indicate standard errors of the means (SEM) ( n = 5). ***, P

    Article Snippet: Briefly, ELISA plates were precoated with bovine mucin (1 mg/ml) at 37°C for 1 h. After blocking with 1% bovine serum albumin (BSA) at 37°C for 1 h, PdCoV S1-NTD (1 μg/ml) was added to the plates and incubated with mucin at 37°C for 1 h. After washes with PBS buffer, the plates were incubated with anti-His6 antibody (Santa Cruz) at 37°C for 1 h. The plates then were washed with PBS and incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG antibody (1:5,000) at 37°C for 1 h. After more washes with PBS, enzymatic reaction was carried out using ELISA substrate (Life Technologies Inc.) and stopped with 1 M H2 SO4 .

    Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay, Incubation, Positive Control, Negative Control, Two Tailed Test

    Preferential binding of PvNod41 to denatured proteins . (A) PvNod41 binding assay. Purified PvNod41 was incubated with either native (N) or denatured (D) proteins pre-immobilized on agarose-beads. After incubation, samples were extensively washed with PBS. PvNod41 that was bound to immobilized proteins on the matrix was recovered by boiling the sample with Laemmli buffer and analyzed by 12% SDS-PAGE and Coomassie Brilliant Blue staining. BSA, Bovine Serum Albumin; α2M, α2-Macroglobulin. (B) Far western blot assay. Bovine serum albumin (BSA) and casein, either native or denatured by boiling were blotted onto nitrocellulose, probed with purified PvNod41, and immunodetected with anti-PvNod41 antiserum as described in the Methods section.

    Journal: BMC Plant Biology

    Article Title: Nodulin 41, a novel late nodulin of common bean with peptidase activity

    doi: 10.1186/1471-2229-11-134

    Figure Lengend Snippet: Preferential binding of PvNod41 to denatured proteins . (A) PvNod41 binding assay. Purified PvNod41 was incubated with either native (N) or denatured (D) proteins pre-immobilized on agarose-beads. After incubation, samples were extensively washed with PBS. PvNod41 that was bound to immobilized proteins on the matrix was recovered by boiling the sample with Laemmli buffer and analyzed by 12% SDS-PAGE and Coomassie Brilliant Blue staining. BSA, Bovine Serum Albumin; α2M, α2-Macroglobulin. (B) Far western blot assay. Bovine serum albumin (BSA) and casein, either native or denatured by boiling were blotted onto nitrocellulose, probed with purified PvNod41, and immunodetected with anti-PvNod41 antiserum as described in the Methods section.

    Article Snippet: For PvNod41 purification, bovine serum albumin (BSA) was immobilized on Affi-Gel 10 Gel (Bio-Rad Laboratories, Hercules, CA, USA) according to the manufacturer's instructions and transferred to a column.

    Techniques: Binding Assay, Purification, Incubation, SDS Page, Staining, Far Western Blot

    Analysis of purified PvNod41 . Protein profile on a Coomassie-stained 12% SDS-PAGE gel of collected fractions obtained during PvNod41 purification. Lane 1, protein marker; lane 2, crude protein extract from root nodules; lane 3, 1 M KCl washing; lane 4, fraction A (elution from the BSA-Affi-Gel 10 Gel column); lane 5, fraction B (flow-through of the chromatography on Affi-Gel Heparin Gel).

    Journal: BMC Plant Biology

    Article Title: Nodulin 41, a novel late nodulin of common bean with peptidase activity

    doi: 10.1186/1471-2229-11-134

    Figure Lengend Snippet: Analysis of purified PvNod41 . Protein profile on a Coomassie-stained 12% SDS-PAGE gel of collected fractions obtained during PvNod41 purification. Lane 1, protein marker; lane 2, crude protein extract from root nodules; lane 3, 1 M KCl washing; lane 4, fraction A (elution from the BSA-Affi-Gel 10 Gel column); lane 5, fraction B (flow-through of the chromatography on Affi-Gel Heparin Gel).

    Article Snippet: For PvNod41 purification, bovine serum albumin (BSA) was immobilized on Affi-Gel 10 Gel (Bio-Rad Laboratories, Hercules, CA, USA) according to the manufacturer's instructions and transferred to a column.

    Techniques: Purification, Staining, SDS Page, Marker, Flow Cytometry, Chromatography

    Mouse hippocampal and cortical neurons (visualized by labeling for PGP 9.5) and nuclei of all cells (visualized by DAPI labeling) after 3 days culture on BSA (A, B, C, and D) merosin (E, F, G, and H) and FN (I, J, K, and L). Lengths of neurites (including axons) are greater on FN than BSA or merosin. Scale bar = 100 μm.

    Journal: Brain Research

    Article Title: Fibronectin supports neurite outgrowth and axonal regeneration of adult brain neurons in vitro

    doi: 10.1016/j.brainres.2012.03.024

    Figure Lengend Snippet: Mouse hippocampal and cortical neurons (visualized by labeling for PGP 9.5) and nuclei of all cells (visualized by DAPI labeling) after 3 days culture on BSA (A, B, C, and D) merosin (E, F, G, and H) and FN (I, J, K, and L). Lengths of neurites (including axons) are greater on FN than BSA or merosin. Scale bar = 100 μm.

    Article Snippet: The resulting pellet was re-suspended in 4 ml of Neurobasal-A with B27 supplement (Invitrogen) containing 2 mM glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin and 250 ng/ml amphotericin B. Aliquots of 0.5 ml of the suspension were added to 4-well culture dishes (Nunc), coated for 1 h with solutions of 20 μg/ml of bovine serum albumin (BSA), laminin (BD Biosciences), merosin (Chemicon) or bovine plasma FN (Sigma), all in PBS.

    Techniques: Labeling