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    Structured Review

    Thermo Fisher sds dtt gel
    Sds Dtt Gel, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Flow Cytometry:

    Article Title: The Effects of Glutaredoxin and Copper Activation Pathways on the Disulfide and Stability of Cu,Zn Superoxide Dismutase *The Effects of Glutaredoxin and Copper Activation Pathways on the Disulfide and Stability of Cu,Zn Superoxide Dismutase * …
    Article Snippet: .. 20 μ l of column flow-through was boiled in SDS-DTT gel loading buffer and clarified by centrifugation prior to analysis by SDS-PAGE on 14% precast gels (Invitrogen). ..

    Centrifugation:

    Article Title: The Effects of Glutaredoxin and Copper Activation Pathways on the Disulfide and Stability of Cu,Zn Superoxide Dismutase *The Effects of Glutaredoxin and Copper Activation Pathways on the Disulfide and Stability of Cu,Zn Superoxide Dismutase * …
    Article Snippet: .. 20 μ l of column flow-through was boiled in SDS-DTT gel loading buffer and clarified by centrifugation prior to analysis by SDS-PAGE on 14% precast gels (Invitrogen). ..

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    Article Title: The Effects of Glutaredoxin and Copper Activation Pathways on the Disulfide and Stability of Cu,Zn Superoxide Dismutase *The Effects of Glutaredoxin and Copper Activation Pathways on the Disulfide and Stability of Cu,Zn Superoxide Dismutase * …
    Article Snippet: .. 20 μ l of column flow-through was boiled in SDS-DTT gel loading buffer and clarified by centrifugation prior to analysis by SDS-PAGE on 14% precast gels (Invitrogen). ..

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    Thermo Fisher protein a sepharose
    Protocol flow chart. In protein cross-linking, recombinant corin protein containing a C-terminal V5 tag (diamond shape) in HEK293 cells was cross-linked with interacting proteins (brown), using dithiobis succinimidyl propionate (DSP). In immunoprecipitation, corin-associated proteins were isolated using an anti-V5 antibody and protein <t>A-Sepharose</t> beads. In proteomic analysis, corin-associated proteins were eluted from the beads under reducing conditions and analyzed by SDS-PAGE followed by in-gel trypsin digestion and LC-MS analysis. The experiments were done in parallel in HEK293 cells expressing corin WT and the mutant N1022Q. Proteins that were differentially associated with corin WT and the mutant N1022Q were selected for further biochemical and cellular studies.
    Protein A Sepharose, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher lasv gpc
    Virus neutralization antibody titers. Day 42 sera from immunized mice was incubated with pseudotyped rVSV-ΔG-NL-GFP. a rVSV-ΔG-NL-GFP was pseudotyped with either RABV-G, <t>LASV-GPC,</t> or EBOV-GP to assay for RABV-G, LASV-GPC, or EBOV-GP NAb titers, respectively. 12.1F, 37.7H, and 25.10c are LASV-GPC neutralizing antibodies used as a positive control for LASV-GPC neutralization 26 . Y -axis in b represents 50% of inhibitory serum dilution (IC 50 ) titers obtained based on the antibody dilution that has 50% infection percentage of infected cells curves obtained in a . All groups achieved high neutralizing titers against RABV-G except for the groups immunized with virus lacking RABV-G: rc-LASSARAB-ΔG and rVSV-coGPC, as expected. Regarding LASV-GPC pseudotyped VSVs, no immunization achieved appreciable amounts of neutralizing antibodies. Neutralization of LASV GPC pseudotyped viruses with 12.1F, 37.7H, and 25.10C had an average IC50 of 1546 ng/ml, 375 ng/ml, and 69 ng/ml, respectively. c Rabies neutralizing titers were calculated by using the IC 50 values of the WHO sera standard (2 IU/ml) serial diluted with rVSV-ΔG-NL-GFP pseudotyped with RABV G. WHO international units/ml (IU/ml) were then calculated using the following formula: (sample IC 50 titer)/(WHO standard IC 50 titer) × 2.0 (WHO IU/ml standard starting dilution). IU/ml from test sera is plotted Y -axis. All error bars represented are the SEM of triplicate values of 5 mice per group. (**** P
    Lasv Gpc, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher hsv 1
    <t>HSV-1</t> ΔgC enters cells via a low-pH-dependent pathway. Vero (A), SK-N-SH (B), IMR-32 (C), HaCaT (E), or HEKa (F) cells were treated with ammonium chloride for 1 h at 37 0 C. Cells were infected with 100 PFU of HSV-1 ΔgC or gCR for 6 h in the continued presence of drug. Normal medium was added, and at 22 h p.i., infectivity was determined by plaque assay. The infectivity of no-drug samples was set to 100%. (D) CHO-HVEM cells were treated with ammonium chloride for 20 min at 37 0 C. HSV-1 gCR or HSV-1 ΔgC was added to cells (MOI of 5) at 37°C in the continued presence of agent. At 6 h p.i., entry was measured as a percentage of beta-galactosidase activity obtained in the absence of ammonium chloride. Data represent means and standard deviations of results from quadruplicate samples and are representative of at least two independent experiments.
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    Thermo Fisher a431
    Expression of nucleolin (cell-surface target protein) and STAT3 in epithelial cancer cell lines. Immunofluorescence using anti-nucleolin antibody showing nucleolin expression in epithelioid carcinoma <t>A431</t> ( A ), HNC line FaDu ( B ); ovarian carcinoma OVCAR3 ( C ). Secondary FITC-conjugated IgG (green) was used. Nuclei were counterstained with DAPI (blue). D - nucleolin expression in membrane (M) and nuclear (N) fractions of cells. Lanes 1,2 - A431, Lanes 3,4 - FaDu, lanes 5,6- HeLa cells. E- electrophoretic mobility shift assay (EMSA) showing STAT3d alone (lane 1,3) and after adding FaDu cell lysate (lane 2,4). STAT3 d was labeled either using Cy5.5 (lanes 1,2) or dual-labeled with Cy5.5 and 800CW(lanes3, 4); F- Western blotting of STAT3 in FaDu cell extract (lane 1) and HeLa extract (lane 2, positive control).
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    Protocol flow chart. In protein cross-linking, recombinant corin protein containing a C-terminal V5 tag (diamond shape) in HEK293 cells was cross-linked with interacting proteins (brown), using dithiobis succinimidyl propionate (DSP). In immunoprecipitation, corin-associated proteins were isolated using an anti-V5 antibody and protein A-Sepharose beads. In proteomic analysis, corin-associated proteins were eluted from the beads under reducing conditions and analyzed by SDS-PAGE followed by in-gel trypsin digestion and LC-MS analysis. The experiments were done in parallel in HEK293 cells expressing corin WT and the mutant N1022Q. Proteins that were differentially associated with corin WT and the mutant N1022Q were selected for further biochemical and cellular studies.

    Journal: Bio-protocol

    Article Title: Cross-linking, Immunoprecipitation and Proteomic Analysis to Identify Interacting Proteins in Cultured Cells

    doi: 10.21769/BioProtoc.3258

    Figure Lengend Snippet: Protocol flow chart. In protein cross-linking, recombinant corin protein containing a C-terminal V5 tag (diamond shape) in HEK293 cells was cross-linked with interacting proteins (brown), using dithiobis succinimidyl propionate (DSP). In immunoprecipitation, corin-associated proteins were isolated using an anti-V5 antibody and protein A-Sepharose beads. In proteomic analysis, corin-associated proteins were eluted from the beads under reducing conditions and analyzed by SDS-PAGE followed by in-gel trypsin digestion and LC-MS analysis. The experiments were done in parallel in HEK293 cells expressing corin WT and the mutant N1022Q. Proteins that were differentially associated with corin WT and the mutant N1022Q were selected for further biochemical and cellular studies.

    Article Snippet: Cell lifters (Corning, catalog number: 3008) Surgical blades (any brand) Pipette tips (any brand) 1.5 ml microcentrifuge tubes (any brand) 150 mm cell culture dishes (Corning, catalog number: 430599) HEK293 cells (ATCC, catalog number: CRL-1573) pcDNA 3.1/V5-His-based plasmids (Thermo Fisher, catalog number: K480001) Dulbecco’s modified Eagle’s medium (DMEM) (Lerner Research Institute Cell Culture Core, catalog number: 11–500) Fetal bovine serum (FBS) (Corning, catalog number: 35–011-CV) Phosphate buffered saline (PBS), 10x (Affymetrix, catalog number: 75889) Dithiobis succinimidyl propionate (DSP) (Thermo Fisher, catalog number: 22585) Dimethyl sulfoxide (DMSO) (Thermo Fisher, catalog number: 24600) Glycine (Research Products International (RPI), catalog number: ) Tris-base (Fisher Scientific, catalog number: 502–13-709) Sodium chloride (RPI, catalog number: S23020) Nonidet P-40 (Affymetrix, catalog number: 19628) Protease inhibitor cocktail (Sigma-Aldrich, catalog number: P8340) Protein assay dye reagent (Bio-Rad, catalog number: 500–0006) Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A9647) Anti-V5 antibody (Thermo Fisher, catalog number: ) Protein A-Sepharose (Thermo Fisher, catalog number: 10–1042) Sodium dodecyl sulfate (SDS) (Fisher Scientific, catalog number: ) SDS-PAGE protein sample buffer (2x) (Bio-Rad, catalog number: 1610737) β-mercaptoethanol (Fisher Scientific, catalog number: BP176–100) Pre-stained protein ladder (Thermo Fisher, catalog number: 26616) Tris-Glycine gel (4–20%) (Thermo Fisher, catalog number: XP04200B0X) Silver staining kit (Thermo Fisher, catalog number: 24600) Ethanol (any brand, HPLC grade) Acetic acid (any brand, HPLC grade) Acetonitrile (any brand, HPLC grade) SilverQuest silver staining kit (Thermo Fisher, catalog number: LC607) Dithiothreitol (DTT) (Thermo Fisher, catalog number: R0861) lodoacetamide (Thermo Fisher, catalog number: ) Trypsin, sequencing grade (Thermo Fisher, catalog number: 90057) Ammonium bicarbonate (Fisher Scientific, catalog number: A643–500) Formic acid (Thermo Fisher, catalog number: 28905) Fugene reagents (Promega, catalog number: E2311) G418 (Teknova, catalog number: G5001) DSP stock solution (see ) Cell lysis buffer (see ) SDS-PAGE buffer (see )

    Techniques: Flow Cytometry, Recombinant, Immunoprecipitation, Isolation, SDS Page, Liquid Chromatography with Mass Spectroscopy, Expressing, Mutagenesis

    Virus neutralization antibody titers. Day 42 sera from immunized mice was incubated with pseudotyped rVSV-ΔG-NL-GFP. a rVSV-ΔG-NL-GFP was pseudotyped with either RABV-G, LASV-GPC, or EBOV-GP to assay for RABV-G, LASV-GPC, or EBOV-GP NAb titers, respectively. 12.1F, 37.7H, and 25.10c are LASV-GPC neutralizing antibodies used as a positive control for LASV-GPC neutralization 26 . Y -axis in b represents 50% of inhibitory serum dilution (IC 50 ) titers obtained based on the antibody dilution that has 50% infection percentage of infected cells curves obtained in a . All groups achieved high neutralizing titers against RABV-G except for the groups immunized with virus lacking RABV-G: rc-LASSARAB-ΔG and rVSV-coGPC, as expected. Regarding LASV-GPC pseudotyped VSVs, no immunization achieved appreciable amounts of neutralizing antibodies. Neutralization of LASV GPC pseudotyped viruses with 12.1F, 37.7H, and 25.10C had an average IC50 of 1546 ng/ml, 375 ng/ml, and 69 ng/ml, respectively. c Rabies neutralizing titers were calculated by using the IC 50 values of the WHO sera standard (2 IU/ml) serial diluted with rVSV-ΔG-NL-GFP pseudotyped with RABV G. WHO international units/ml (IU/ml) were then calculated using the following formula: (sample IC 50 titer)/(WHO standard IC 50 titer) × 2.0 (WHO IU/ml standard starting dilution). IU/ml from test sera is plotted Y -axis. All error bars represented are the SEM of triplicate values of 5 mice per group. (**** P

    Journal: Nature Communications

    Article Title: Non-neutralizing antibodies elicited by recombinant Lassa–Rabies vaccine are critical for protection against Lassa fever

    doi: 10.1038/s41467-018-06741-w

    Figure Lengend Snippet: Virus neutralization antibody titers. Day 42 sera from immunized mice was incubated with pseudotyped rVSV-ΔG-NL-GFP. a rVSV-ΔG-NL-GFP was pseudotyped with either RABV-G, LASV-GPC, or EBOV-GP to assay for RABV-G, LASV-GPC, or EBOV-GP NAb titers, respectively. 12.1F, 37.7H, and 25.10c are LASV-GPC neutralizing antibodies used as a positive control for LASV-GPC neutralization 26 . Y -axis in b represents 50% of inhibitory serum dilution (IC 50 ) titers obtained based on the antibody dilution that has 50% infection percentage of infected cells curves obtained in a . All groups achieved high neutralizing titers against RABV-G except for the groups immunized with virus lacking RABV-G: rc-LASSARAB-ΔG and rVSV-coGPC, as expected. Regarding LASV-GPC pseudotyped VSVs, no immunization achieved appreciable amounts of neutralizing antibodies. Neutralization of LASV GPC pseudotyped viruses with 12.1F, 37.7H, and 25.10C had an average IC50 of 1546 ng/ml, 375 ng/ml, and 69 ng/ml, respectively. c Rabies neutralizing titers were calculated by using the IC 50 values of the WHO sera standard (2 IU/ml) serial diluted with rVSV-ΔG-NL-GFP pseudotyped with RABV G. WHO international units/ml (IU/ml) were then calculated using the following formula: (sample IC 50 titer)/(WHO standard IC 50 titer) × 2.0 (WHO IU/ml standard starting dilution). IU/ml from test sera is plotted Y -axis. All error bars represented are the SEM of triplicate values of 5 mice per group. (**** P

    Article Snippet: Viruses and ELISA antigen characterization Virus particles and purified LASV GPC were denatured with Urea Sample Buffer (125 mM Tris-HCl [pH 6.8], 8 M urea, 4% sodium dodecyl sulfate, 50 mM dithiothreitol, 0.02% bromophenol blue) at 95 °C for 5 min. 2 μg of protein was resolved on a 10% SDS–polyacrylamide gel and stained O/N with SYPRO Ruby (Thermofisher) for total protein analysis.

    Techniques: Neutralization, Mouse Assay, Incubation, Gel Permeation Chromatography, Positive Control, Infection

    Evaluation of in vivo relevance of non-NAbs LASV GPC specific antibodies induced by LASSARAB + GLA-SE vaccination. a 8- to 10-week-old Balb/c (WT) or Balb/c with Fcγ chain KO (Fcγ −/− ) female mice were immunized with 10 µg of inactivated particles of either LASSARAB or FILORAB1 (mock control) on day 0 and boosted on day 28. All four groups in total were adjuvanted with 5 µg of GLA in a 2% SE with each vaccination. One day before exposure (day 41) animals were injected with 1.25 mg of anti-Ifnar mAb (MAR1-5A3, Leinco technologies) through intra-peritoneal injection (IP). On day 42, mice were exposed to 10 4 rVSV-GPC virus IP and general health (weights and clinical observation) was recorded until endpoint criteria were reached or end of study (supplemental). b Survival curves post-exposure of rVSV-GPC. Significance is compared between the WT LASSARAB vaccinated and the Fcγ −/− vaccinated using the log-rank (Mantel–Cox) test. c Pre-exposure total IgG titers anti LASV GPC were measured by ELISA on day 35 post-prime and ELISA curves were plotted according to OD490 reading value ( Y -axis) and serum dilution ( X -axis). On the right, EC 50 (half maximal effective concentration) of serum dilution of both LASSARAB groups (WT and Fcγ −/− ) is plotted on Y -axis on a log scale; statistical significance was calculated using one-way ANOVA. d Virus neutralization assay using pseudotyped VSV-GFP-NanoLuc with LASV GPC. On the right, percentage of cells infected is plotted against the serum dilution (survivors on day 14 post-exposure) of each respective group. On the right, the IC 50 (half maximal inhibitory concentration) of serum dilution is plotted individually and significance was calculated using one-way ANOVA. Error bars represent Standard Error Mean (SEM) and include all mice ( n = 10 per group [WT and KO] in LASSARAB and n = 5 per group [WT and KO] in FILORAB1 control) in pre-challenge and survivor mice in post-challenge. (**** P

    Journal: Nature Communications

    Article Title: Non-neutralizing antibodies elicited by recombinant Lassa–Rabies vaccine are critical for protection against Lassa fever

    doi: 10.1038/s41467-018-06741-w

    Figure Lengend Snippet: Evaluation of in vivo relevance of non-NAbs LASV GPC specific antibodies induced by LASSARAB + GLA-SE vaccination. a 8- to 10-week-old Balb/c (WT) or Balb/c with Fcγ chain KO (Fcγ −/− ) female mice were immunized with 10 µg of inactivated particles of either LASSARAB or FILORAB1 (mock control) on day 0 and boosted on day 28. All four groups in total were adjuvanted with 5 µg of GLA in a 2% SE with each vaccination. One day before exposure (day 41) animals were injected with 1.25 mg of anti-Ifnar mAb (MAR1-5A3, Leinco technologies) through intra-peritoneal injection (IP). On day 42, mice were exposed to 10 4 rVSV-GPC virus IP and general health (weights and clinical observation) was recorded until endpoint criteria were reached or end of study (supplemental). b Survival curves post-exposure of rVSV-GPC. Significance is compared between the WT LASSARAB vaccinated and the Fcγ −/− vaccinated using the log-rank (Mantel–Cox) test. c Pre-exposure total IgG titers anti LASV GPC were measured by ELISA on day 35 post-prime and ELISA curves were plotted according to OD490 reading value ( Y -axis) and serum dilution ( X -axis). On the right, EC 50 (half maximal effective concentration) of serum dilution of both LASSARAB groups (WT and Fcγ −/− ) is plotted on Y -axis on a log scale; statistical significance was calculated using one-way ANOVA. d Virus neutralization assay using pseudotyped VSV-GFP-NanoLuc with LASV GPC. On the right, percentage of cells infected is plotted against the serum dilution (survivors on day 14 post-exposure) of each respective group. On the right, the IC 50 (half maximal inhibitory concentration) of serum dilution is plotted individually and significance was calculated using one-way ANOVA. Error bars represent Standard Error Mean (SEM) and include all mice ( n = 10 per group [WT and KO] in LASSARAB and n = 5 per group [WT and KO] in FILORAB1 control) in pre-challenge and survivor mice in post-challenge. (**** P

    Article Snippet: Viruses and ELISA antigen characterization Virus particles and purified LASV GPC were denatured with Urea Sample Buffer (125 mM Tris-HCl [pH 6.8], 8 M urea, 4% sodium dodecyl sulfate, 50 mM dithiothreitol, 0.02% bromophenol blue) at 95 °C for 5 min. 2 μg of protein was resolved on a 10% SDS–polyacrylamide gel and stained O/N with SYPRO Ruby (Thermofisher) for total protein analysis.

    Techniques: In Vivo, Gel Permeation Chromatography, Mouse Assay, Injection, Enzyme-linked Immunosorbent Assay, Concentration Assay, Neutralization, Infection

    LASV challenge of outbred Hartley guinea pigs immunized with several vaccine candidates and control. a Guinea pigs were immunized with either two replication competent vaccines: rVSV-GPC (positive control for survival) and LASSARAB replication-competent at 10 6 ffu by intraperitoneal injection (IP); or inactivated LASSARAB+GLA-SE with different immunization schedules: day −58 (LASSARAB+GLA-SE (1)), day −58, day −51 (LASSARAB+GLA-SE (2)) and day −58, day −51, and day −30 (LASSARAB+GLA-SE (3)). RabAvert was used as mock immunization (negative control). b Survival curves post IP exposure with 10 4 pfu guinea pig-adapted LASV Josiah strain. Statistical significance is compared against Rabvert group using log-rank (Mantel–Cox) test. c Heat plot representing the clinical score information. X -axis represents days' post-challenge and Y -axis represents the individual animal number. d Terminal viremia was plotted using LASV RNA copies/ml in Y -axis. Statistical significance was calculated using Kruskal–Wallis one-way ANOVA (not significant). e LASV neutralizing antibody titers is reported as the IC 50 (half maximal inhibitory concentration) of serum dilution. The human mAbs 25.10C, 12.1F, and 37.7H 25 , 26 were used as positive LASV neutralization controls. f Pre-challenge titers of LASV GPC specific IgG were performed on sera collected on day −15 prior to challenge by ELISA with LASV GPC antigen and the EC 50 (50% effective concentration) of serum dilution was plotted in the Y -axis. Statistical significance (compared to the RabAvert group) was calculated by using one-way ANOVA (post-hoc test Tukey Honest Significant Difference Test). g Post-challenge titers of LASV GPC-specific IgG was performed on sera collected on terminal bleeding of both succumbed animals and survivors (day 50 post challenge) and the EC 50 of serum dilution is plotted on the Y -axis. Statistical significance reported between survivors and succumbed in e , g was determined by using two-way ANOVA. All error bars represented are the standard error mean (SEM) of 10 animals per group (in triplicates). (**** P

    Journal: Nature Communications

    Article Title: Non-neutralizing antibodies elicited by recombinant Lassa–Rabies vaccine are critical for protection against Lassa fever

    doi: 10.1038/s41467-018-06741-w

    Figure Lengend Snippet: LASV challenge of outbred Hartley guinea pigs immunized with several vaccine candidates and control. a Guinea pigs were immunized with either two replication competent vaccines: rVSV-GPC (positive control for survival) and LASSARAB replication-competent at 10 6 ffu by intraperitoneal injection (IP); or inactivated LASSARAB+GLA-SE with different immunization schedules: day −58 (LASSARAB+GLA-SE (1)), day −58, day −51 (LASSARAB+GLA-SE (2)) and day −58, day −51, and day −30 (LASSARAB+GLA-SE (3)). RabAvert was used as mock immunization (negative control). b Survival curves post IP exposure with 10 4 pfu guinea pig-adapted LASV Josiah strain. Statistical significance is compared against Rabvert group using log-rank (Mantel–Cox) test. c Heat plot representing the clinical score information. X -axis represents days' post-challenge and Y -axis represents the individual animal number. d Terminal viremia was plotted using LASV RNA copies/ml in Y -axis. Statistical significance was calculated using Kruskal–Wallis one-way ANOVA (not significant). e LASV neutralizing antibody titers is reported as the IC 50 (half maximal inhibitory concentration) of serum dilution. The human mAbs 25.10C, 12.1F, and 37.7H 25 , 26 were used as positive LASV neutralization controls. f Pre-challenge titers of LASV GPC specific IgG were performed on sera collected on day −15 prior to challenge by ELISA with LASV GPC antigen and the EC 50 (50% effective concentration) of serum dilution was plotted in the Y -axis. Statistical significance (compared to the RabAvert group) was calculated by using one-way ANOVA (post-hoc test Tukey Honest Significant Difference Test). g Post-challenge titers of LASV GPC-specific IgG was performed on sera collected on terminal bleeding of both succumbed animals and survivors (day 50 post challenge) and the EC 50 of serum dilution is plotted on the Y -axis. Statistical significance reported between survivors and succumbed in e , g was determined by using two-way ANOVA. All error bars represented are the standard error mean (SEM) of 10 animals per group (in triplicates). (**** P

    Article Snippet: Viruses and ELISA antigen characterization Virus particles and purified LASV GPC were denatured with Urea Sample Buffer (125 mM Tris-HCl [pH 6.8], 8 M urea, 4% sodium dodecyl sulfate, 50 mM dithiothreitol, 0.02% bromophenol blue) at 95 °C for 5 min. 2 μg of protein was resolved on a 10% SDS–polyacrylamide gel and stained O/N with SYPRO Ruby (Thermofisher) for total protein analysis.

    Techniques: Gel Permeation Chromatography, Positive Control, Injection, Negative Control, Concentration Assay, Neutralization, Enzyme-linked Immunosorbent Assay

    Evaluation of antibody effector cell functions mediated by murine NK and macrophage cells against 3T3 expressing LASV GPC. Day 42 sera from immunized mice was incubated with 3T3-LASV cells and 30 min either murine NK or macrophage cells were added and results were analyzed 4 h later by either flow cytometry (Supplementary Fig. 3 and a – d ) or confocal microscopy ( e ). Purified murine C57BL/6 NK cells ( a ) or IC-21 macrophages ( d ) were added at different Target:Effector cell ratios (T:E) with target cells incubated with either LASSARAB sera (yellow), FILORAB1 sera (gray) or no sera (pink). The Y -axis represents the percentage of cellular cytotoxicity based on GFP + /PI + cells (gating strategy and flow plots in Supplementary Fig. 3 ). b To determine which antibody isotype class is important for ADCC, NK cells were added at 1:5 T:E and incubated with either unprocessed sera (sera 1:100 condition), purified IgG (20 μg/ml), or IgG impoverished sera (1:5 dilution) from LASSARAB and FILORAB1 immunized mice. The Y axis represents cytotoxicity fold change of LASSARAB sera or IgG compared to FILORAB1 sera or IgG with same respective conditions. Anti-CD16/32 (Fcγ-RII/III) was also added at 25 μg/ml to confirm that FcγR blockade reduces ADCC activity. c , e , f To analyze ADCP J774.A1 macrophages were added at 1:5T:E or 1:1T:E (confocal) to 3T3-LASV cells incubated with either LASSARAB sera ( c , e ) or FILORAB1 sera ( c , f ). In c anti-CD16.2 (Fcγ-RIV), anti-CD16/32 (Fcγ-RII/III), and anti-CD64 (Fcγ-RI) were added at 25 μg/ml to check the effect of different FcγR blockade on ADCP activity. All error bars are the SEM of at least three independent experiments executed with duplicates. All statistical significance represented was performed through either a one- or two-way ANOVA and using a post-hoc analysis Tukey Honest Significant Difference test. (**** P

    Journal: Nature Communications

    Article Title: Non-neutralizing antibodies elicited by recombinant Lassa–Rabies vaccine are critical for protection against Lassa fever

    doi: 10.1038/s41467-018-06741-w

    Figure Lengend Snippet: Evaluation of antibody effector cell functions mediated by murine NK and macrophage cells against 3T3 expressing LASV GPC. Day 42 sera from immunized mice was incubated with 3T3-LASV cells and 30 min either murine NK or macrophage cells were added and results were analyzed 4 h later by either flow cytometry (Supplementary Fig. 3 and a – d ) or confocal microscopy ( e ). Purified murine C57BL/6 NK cells ( a ) or IC-21 macrophages ( d ) were added at different Target:Effector cell ratios (T:E) with target cells incubated with either LASSARAB sera (yellow), FILORAB1 sera (gray) or no sera (pink). The Y -axis represents the percentage of cellular cytotoxicity based on GFP + /PI + cells (gating strategy and flow plots in Supplementary Fig. 3 ). b To determine which antibody isotype class is important for ADCC, NK cells were added at 1:5 T:E and incubated with either unprocessed sera (sera 1:100 condition), purified IgG (20 μg/ml), or IgG impoverished sera (1:5 dilution) from LASSARAB and FILORAB1 immunized mice. The Y axis represents cytotoxicity fold change of LASSARAB sera or IgG compared to FILORAB1 sera or IgG with same respective conditions. Anti-CD16/32 (Fcγ-RII/III) was also added at 25 μg/ml to confirm that FcγR blockade reduces ADCC activity. c , e , f To analyze ADCP J774.A1 macrophages were added at 1:5T:E or 1:1T:E (confocal) to 3T3-LASV cells incubated with either LASSARAB sera ( c , e ) or FILORAB1 sera ( c , f ). In c anti-CD16.2 (Fcγ-RIV), anti-CD16/32 (Fcγ-RII/III), and anti-CD64 (Fcγ-RI) were added at 25 μg/ml to check the effect of different FcγR blockade on ADCP activity. All error bars are the SEM of at least three independent experiments executed with duplicates. All statistical significance represented was performed through either a one- or two-way ANOVA and using a post-hoc analysis Tukey Honest Significant Difference test. (**** P

    Article Snippet: Viruses and ELISA antigen characterization Virus particles and purified LASV GPC were denatured with Urea Sample Buffer (125 mM Tris-HCl [pH 6.8], 8 M urea, 4% sodium dodecyl sulfate, 50 mM dithiothreitol, 0.02% bromophenol blue) at 95 °C for 5 min. 2 μg of protein was resolved on a 10% SDS–polyacrylamide gel and stained O/N with SYPRO Ruby (Thermofisher) for total protein analysis.

    Techniques: Expressing, Gel Permeation Chromatography, Mouse Assay, Incubation, Flow Cytometry, Cytometry, Confocal Microscopy, Purification, Activity Assay

    Diagram of vaccine constructs and controls. BNSP333 is the parental vector and FILORAB1, the control used, is based on BNSP333 with a codon-optimized Zaire Ebola Virus Glycoprotein (EBOV GP) inserted between N and P through the BsiWI and NheI restriction digest sites. LASSARAB was generated in a similar manner as FILORAB1, from BNSP333 by cloning a codon-optimized version of Lassa virus glycoprotein (LASV GPC) in the BsiWI and NheI restriction digest sites. LASSARAB-ΔG was further generated from LASSARAB by removing the native rabies glycoprotein (G) by using the restriction digest sites SmaI and PacI. rVSV-GPC was generated by replacing the native VSV glycoprotein (G) by LASV GPC at the same sites. rVSV-GPC was created to be used as a control vector and as a scaffold to produce a native LASV GPC antigen for ELISAs (see Methods section)

    Journal: Nature Communications

    Article Title: Non-neutralizing antibodies elicited by recombinant Lassa–Rabies vaccine are critical for protection against Lassa fever

    doi: 10.1038/s41467-018-06741-w

    Figure Lengend Snippet: Diagram of vaccine constructs and controls. BNSP333 is the parental vector and FILORAB1, the control used, is based on BNSP333 with a codon-optimized Zaire Ebola Virus Glycoprotein (EBOV GP) inserted between N and P through the BsiWI and NheI restriction digest sites. LASSARAB was generated in a similar manner as FILORAB1, from BNSP333 by cloning a codon-optimized version of Lassa virus glycoprotein (LASV GPC) in the BsiWI and NheI restriction digest sites. LASSARAB-ΔG was further generated from LASSARAB by removing the native rabies glycoprotein (G) by using the restriction digest sites SmaI and PacI. rVSV-GPC was generated by replacing the native VSV glycoprotein (G) by LASV GPC at the same sites. rVSV-GPC was created to be used as a control vector and as a scaffold to produce a native LASV GPC antigen for ELISAs (see Methods section)

    Article Snippet: Viruses and ELISA antigen characterization Virus particles and purified LASV GPC were denatured with Urea Sample Buffer (125 mM Tris-HCl [pH 6.8], 8 M urea, 4% sodium dodecyl sulfate, 50 mM dithiothreitol, 0.02% bromophenol blue) at 95 °C for 5 min. 2 μg of protein was resolved on a 10% SDS–polyacrylamide gel and stained O/N with SYPRO Ruby (Thermofisher) for total protein analysis.

    Techniques: Construct, Plasmid Preparation, Generated, Clone Assay, Gel Permeation Chromatography

    Evaluation of LASSARAB and LASSARAB-ΔG vectors in cell culture and inactivated virion characterization. a LASSARAB, FILORAB1 and uninfected VERO cells were probed for LASV-GPC and RABV-G expression with 37.7H anti-LASV human mAb and 1C5 anti-RABV G mouse mAb and analyzed by flow cytometry 48 h post infection. b VERO cells were infected at a MOI of 0.1 with 4 viruses: FILORAB1, LASSARAB, LASSARAB-ΔG, and rVSV-coGPC. 48 h later (24 h for VSV based vectors) cell surface expression of LASV glycoprotein (GPC), in red, and RABV glycoprotein (G), in green, was probed by a α-LASV GPC rabbit polyclonal and a α-RABV G human 4C12 monoclonal, respectively. In LASSARAB infected cells, yellow is observed as the superimposition of LASV GPC surface expression with RABV G. The bar indicates 12 μm. c VERO CCL-81 cells were infected with a MOI of 0.01 and media supernatant was collected at 0, 24, 48, 72, and 96 h. Virus titers were measured through foci-forming assay (in Y -axis) and plotted through time ( X -axis). d , e LASSARAB and FILORAB1 virions were concentrated through TFF and sucrose purified through ultra-centrifugation. Pellets were resuspended in PBS, BPL inactivated at 1:2000 for 24 h, and 2 μg of each was loaded in a denaturing 10% SDS-PAGE gel. In d SYPRO Ruby staining was used. e , f LASV GPC incorporation in LASSARAB particles was confirmed by western Blot with either an anti-LASV GP2 rabbit polyclonal (upper panel) and anti-GPC/GP1/GP2 guinea pig survivor serum (lower panel). Uncropped versions are available in supplementary figures

    Journal: Nature Communications

    Article Title: Non-neutralizing antibodies elicited by recombinant Lassa–Rabies vaccine are critical for protection against Lassa fever

    doi: 10.1038/s41467-018-06741-w

    Figure Lengend Snippet: Evaluation of LASSARAB and LASSARAB-ΔG vectors in cell culture and inactivated virion characterization. a LASSARAB, FILORAB1 and uninfected VERO cells were probed for LASV-GPC and RABV-G expression with 37.7H anti-LASV human mAb and 1C5 anti-RABV G mouse mAb and analyzed by flow cytometry 48 h post infection. b VERO cells were infected at a MOI of 0.1 with 4 viruses: FILORAB1, LASSARAB, LASSARAB-ΔG, and rVSV-coGPC. 48 h later (24 h for VSV based vectors) cell surface expression of LASV glycoprotein (GPC), in red, and RABV glycoprotein (G), in green, was probed by a α-LASV GPC rabbit polyclonal and a α-RABV G human 4C12 monoclonal, respectively. In LASSARAB infected cells, yellow is observed as the superimposition of LASV GPC surface expression with RABV G. The bar indicates 12 μm. c VERO CCL-81 cells were infected with a MOI of 0.01 and media supernatant was collected at 0, 24, 48, 72, and 96 h. Virus titers were measured through foci-forming assay (in Y -axis) and plotted through time ( X -axis). d , e LASSARAB and FILORAB1 virions were concentrated through TFF and sucrose purified through ultra-centrifugation. Pellets were resuspended in PBS, BPL inactivated at 1:2000 for 24 h, and 2 μg of each was loaded in a denaturing 10% SDS-PAGE gel. In d SYPRO Ruby staining was used. e , f LASV GPC incorporation in LASSARAB particles was confirmed by western Blot with either an anti-LASV GP2 rabbit polyclonal (upper panel) and anti-GPC/GP1/GP2 guinea pig survivor serum (lower panel). Uncropped versions are available in supplementary figures

    Article Snippet: Viruses and ELISA antigen characterization Virus particles and purified LASV GPC were denatured with Urea Sample Buffer (125 mM Tris-HCl [pH 6.8], 8 M urea, 4% sodium dodecyl sulfate, 50 mM dithiothreitol, 0.02% bromophenol blue) at 95 °C for 5 min. 2 μg of protein was resolved on a 10% SDS–polyacrylamide gel and stained O/N with SYPRO Ruby (Thermofisher) for total protein analysis.

    Techniques: Cell Culture, Gel Permeation Chromatography, Expressing, Flow Cytometry, Cytometry, Infection, Purification, Centrifugation, SDS Page, Staining, Western Blot

    Analysis of the humoral response towards Lassa virus glycoprotein. C57BL/6 mice were immunized IM in the gastrocnemius muscle with either 10 μg of β-propiolactone inactivated viral particles in PBS or adjuvanted with 5 μg of GLA, a TLR-4 agonist formulated in 2% of stable emulsion (SE); LASSARAB+GLA-SE, LASSARAB, FILORAB1 groups) and boosted two times with the same amount on day 7 and 28 ( a ). Immunizations with replication-competent viruses were executed with a single time inoculation of 10 6 ffu or pfu virus IM in the gastrocnemius (rc-LASSARAB; rc-FILORAB1 groups and rVSV-GPC). b The EC 50 values (obtained from the 4PL regression ELISA curve) of the total IgG titers against LASV GPC are plotted since day 0 until day 42. Error bars are representative of the standard error mean (SEM) and is calculated from 15 mice per group. Statistical significance was calculated by using 2-way ANOVA–post-hoc Tukey’s Honest Significant Difference Test. c ELISA of total IgG against LASV GPC of all day 42 groups are shown for all immunized groups. ELISA curves are generated from 4PL regression. Error bars are representative of the SEM of OD 490 values (five mice per group, in triplicates). d Day 35 EC 50 antibody titer of IgG sub-isotype (IgG2c and IgG1) against LASV GPC of sera from LASSARAB+GLA-SE and LASSARAB group was analyzed. Error bars are the SEM of a total of five mice per group and statistical significance by 2-way ANOVA (post-hoc Tukey’s Honest Significant Difference Test). e The ratios of the respective EC 50 antibody titers IgG1/IgG2c are plotted and the F test was applied to check for variance difference ( p

    Journal: Nature Communications

    Article Title: Non-neutralizing antibodies elicited by recombinant Lassa–Rabies vaccine are critical for protection against Lassa fever

    doi: 10.1038/s41467-018-06741-w

    Figure Lengend Snippet: Analysis of the humoral response towards Lassa virus glycoprotein. C57BL/6 mice were immunized IM in the gastrocnemius muscle with either 10 μg of β-propiolactone inactivated viral particles in PBS or adjuvanted with 5 μg of GLA, a TLR-4 agonist formulated in 2% of stable emulsion (SE); LASSARAB+GLA-SE, LASSARAB, FILORAB1 groups) and boosted two times with the same amount on day 7 and 28 ( a ). Immunizations with replication-competent viruses were executed with a single time inoculation of 10 6 ffu or pfu virus IM in the gastrocnemius (rc-LASSARAB; rc-FILORAB1 groups and rVSV-GPC). b The EC 50 values (obtained from the 4PL regression ELISA curve) of the total IgG titers against LASV GPC are plotted since day 0 until day 42. Error bars are representative of the standard error mean (SEM) and is calculated from 15 mice per group. Statistical significance was calculated by using 2-way ANOVA–post-hoc Tukey’s Honest Significant Difference Test. c ELISA of total IgG against LASV GPC of all day 42 groups are shown for all immunized groups. ELISA curves are generated from 4PL regression. Error bars are representative of the SEM of OD 490 values (five mice per group, in triplicates). d Day 35 EC 50 antibody titer of IgG sub-isotype (IgG2c and IgG1) against LASV GPC of sera from LASSARAB+GLA-SE and LASSARAB group was analyzed. Error bars are the SEM of a total of five mice per group and statistical significance by 2-way ANOVA (post-hoc Tukey’s Honest Significant Difference Test). e The ratios of the respective EC 50 antibody titers IgG1/IgG2c are plotted and the F test was applied to check for variance difference ( p

    Article Snippet: Viruses and ELISA antigen characterization Virus particles and purified LASV GPC were denatured with Urea Sample Buffer (125 mM Tris-HCl [pH 6.8], 8 M urea, 4% sodium dodecyl sulfate, 50 mM dithiothreitol, 0.02% bromophenol blue) at 95 °C for 5 min. 2 μg of protein was resolved on a 10% SDS–polyacrylamide gel and stained O/N with SYPRO Ruby (Thermofisher) for total protein analysis.

    Techniques: Mouse Assay, Gel Permeation Chromatography, Enzyme-linked Immunosorbent Assay, Generated

    HSV-1 ΔgC enters cells via a low-pH-dependent pathway. Vero (A), SK-N-SH (B), IMR-32 (C), HaCaT (E), or HEKa (F) cells were treated with ammonium chloride for 1 h at 37 0 C. Cells were infected with 100 PFU of HSV-1 ΔgC or gCR for 6 h in the continued presence of drug. Normal medium was added, and at 22 h p.i., infectivity was determined by plaque assay. The infectivity of no-drug samples was set to 100%. (D) CHO-HVEM cells were treated with ammonium chloride for 20 min at 37 0 C. HSV-1 gCR or HSV-1 ΔgC was added to cells (MOI of 5) at 37°C in the continued presence of agent. At 6 h p.i., entry was measured as a percentage of beta-galactosidase activity obtained in the absence of ammonium chloride. Data represent means and standard deviations of results from quadruplicate samples and are representative of at least two independent experiments.

    Journal: mSphere

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

    doi: 10.1128/mSphere.00826-19

    Figure Lengend Snippet: HSV-1 ΔgC enters cells via a low-pH-dependent pathway. Vero (A), SK-N-SH (B), IMR-32 (C), HaCaT (E), or HEKa (F) cells were treated with ammonium chloride for 1 h at 37 0 C. Cells were infected with 100 PFU of HSV-1 ΔgC or gCR for 6 h in the continued presence of drug. Normal medium was added, and at 22 h p.i., infectivity was determined by plaque assay. The infectivity of no-drug samples was set to 100%. (D) CHO-HVEM cells were treated with ammonium chloride for 20 min at 37 0 C. HSV-1 gCR or HSV-1 ΔgC was added to cells (MOI of 5) at 37°C in the continued presence of agent. At 6 h p.i., entry was measured as a percentage of beta-galactosidase activity obtained in the absence of ammonium chloride. Data represent means and standard deviations of results from quadruplicate samples and are representative of at least two independent experiments.

    Article Snippet: HSV-1 was boiled in Laemmli buffer containing 200 mM dithiothreitol for 5 min. Proteins were separated by SDS-PAGE on Tris-glycine gels (Thermo Fisher Scientific).

    Techniques: Infection, Plaque Assay, Activity Assay

    Efficiency of gC-negative HSV-1 infection of human cells that support pH-neutral or low-pH entry. (A and B) HSV-1 gCR (A) and ΔgC (B) titers were determined on SK-N-SH or HaCaT cells by plaque assay. (C and D) Attempt to restore infectivity of HSV-1 lacking gC by providing gC in the cell. Equivalent inocula of HSV-1 gCR (C) or ΔgC (D) were added to SK-N-SH or HaCaT cells transfected with empty vector or gC or gD plasmids at 37 0 C. At 18 to 24 h p.i., titers were determined by plaque assay. Data represent means of results from three independent experiments performed in at least triplicate with standard deviations. **, P

    Journal: mSphere

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

    doi: 10.1128/mSphere.00826-19

    Figure Lengend Snippet: Efficiency of gC-negative HSV-1 infection of human cells that support pH-neutral or low-pH entry. (A and B) HSV-1 gCR (A) and ΔgC (B) titers were determined on SK-N-SH or HaCaT cells by plaque assay. (C and D) Attempt to restore infectivity of HSV-1 lacking gC by providing gC in the cell. Equivalent inocula of HSV-1 gCR (C) or ΔgC (D) were added to SK-N-SH or HaCaT cells transfected with empty vector or gC or gD plasmids at 37 0 C. At 18 to 24 h p.i., titers were determined by plaque assay. Data represent means of results from three independent experiments performed in at least triplicate with standard deviations. **, P

    Article Snippet: HSV-1 was boiled in Laemmli buffer containing 200 mM dithiothreitol for 5 min. Proteins were separated by SDS-PAGE on Tris-glycine gels (Thermo Fisher Scientific).

    Techniques: Infection, Plaque Assay, Transfection, Plasmid Preparation

    The pH threshold of reversibility of gB conformational changes. HSV-1 ΔgC or gCR was treated with pH 7.3 or 5.0 and incubated for 10 min. The pHs of the pH 5-treated samples were increased to the indicated levels for an additional 10 min. Samples were directly blotted to nitrocellulose and probed at neutral pH with gB MAb H126 (A), H1781 (B), SS144 (C), or H1817 (D). MAb reactivity was quantitated, with the pH 7.3-treated sample set to 100%. Data shown are representative of results from two independent experiments.

    Journal: mSphere

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

    doi: 10.1128/mSphere.00826-19

    Figure Lengend Snippet: The pH threshold of reversibility of gB conformational changes. HSV-1 ΔgC or gCR was treated with pH 7.3 or 5.0 and incubated for 10 min. The pHs of the pH 5-treated samples were increased to the indicated levels for an additional 10 min. Samples were directly blotted to nitrocellulose and probed at neutral pH with gB MAb H126 (A), H1781 (B), SS144 (C), or H1817 (D). MAb reactivity was quantitated, with the pH 7.3-treated sample set to 100%. Data shown are representative of results from two independent experiments.

    Article Snippet: HSV-1 was boiled in Laemmli buffer containing 200 mM dithiothreitol for 5 min. Proteins were separated by SDS-PAGE on Tris-glycine gels (Thermo Fisher Scientific).

    Techniques: Incubation

    gC contributes to rapid HSV penetration following endocytosis. HSV-1 ΔgC or gCR was bound to CHO-HVEM (A) or HEKa (B) cells for 1 h at 4°C (MOI of 8). Following a shift to 37 0 C, extracellular virions were subjected to citrate inactivation at the indicated times p.i. Titers of freeze-thaw cell lysates were determined on Vero cells as an indication of infectious, enveloped, intracellular particles. This allows monitoring of viral trafficking and penetration over time. Peak recovery titers were set to 100%. Data are representative of results from at least two independent experiments.

    Journal: mSphere

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

    doi: 10.1128/mSphere.00826-19

    Figure Lengend Snippet: gC contributes to rapid HSV penetration following endocytosis. HSV-1 ΔgC or gCR was bound to CHO-HVEM (A) or HEKa (B) cells for 1 h at 4°C (MOI of 8). Following a shift to 37 0 C, extracellular virions were subjected to citrate inactivation at the indicated times p.i. Titers of freeze-thaw cell lysates were determined on Vero cells as an indication of infectious, enveloped, intracellular particles. This allows monitoring of viral trafficking and penetration over time. Peak recovery titers were set to 100%. Data are representative of results from at least two independent experiments.

    Article Snippet: HSV-1 was boiled in Laemmli buffer containing 200 mM dithiothreitol for 5 min. Proteins were separated by SDS-PAGE on Tris-glycine gels (Thermo Fisher Scientific).

    Techniques:

    Low-pH-induced changes in gB oligomer are independent of gC, as is the reversibility of those changes. (A) HSV-1 gCR or ΔgC was treated with a range of pHs for 10 min. Samples were directly blotted to nitrocellulose and probed at neutral pH with gB oligomer-specific MAb DL16. Reactivity was quantitated, and the level seen with the pH 7.3 sample was set as 100%. (B) HSV-1 gCR or ΔgC was treated with a range of pHs for 10 min. SDS (1%) was added, and reactions were added to “native” PAGE sample buffer. Unheated samples were resolved by 8% SDS-PAGE and Western blot for HSV-1 gB. (C) HSV-1 gCR or ΔgC was treated with pH 5 for 10 min and then blotted directly to nitrocellulose or first neutralized back to pH 7.3 for 10 min and then blotted. Blots were probed at neutral pH with anti-gB MAb DL16. (D) HSV-1 gCR or ΔgC was treated with pH 5 for 10 min. One set of samples was neutralized back to pH 7.3. SDS (1%) was added, and samples were processed as described for panel B. Molecular size markers are indicated in kilodaltons at the right. Data shown are representative of results from at least two independent experiments.

    Journal: mSphere

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

    doi: 10.1128/mSphere.00826-19

    Figure Lengend Snippet: Low-pH-induced changes in gB oligomer are independent of gC, as is the reversibility of those changes. (A) HSV-1 gCR or ΔgC was treated with a range of pHs for 10 min. Samples were directly blotted to nitrocellulose and probed at neutral pH with gB oligomer-specific MAb DL16. Reactivity was quantitated, and the level seen with the pH 7.3 sample was set as 100%. (B) HSV-1 gCR or ΔgC was treated with a range of pHs for 10 min. SDS (1%) was added, and reactions were added to “native” PAGE sample buffer. Unheated samples were resolved by 8% SDS-PAGE and Western blot for HSV-1 gB. (C) HSV-1 gCR or ΔgC was treated with pH 5 for 10 min and then blotted directly to nitrocellulose or first neutralized back to pH 7.3 for 10 min and then blotted. Blots were probed at neutral pH with anti-gB MAb DL16. (D) HSV-1 gCR or ΔgC was treated with pH 5 for 10 min. One set of samples was neutralized back to pH 7.3. SDS (1%) was added, and samples were processed as described for panel B. Molecular size markers are indicated in kilodaltons at the right. Data shown are representative of results from at least two independent experiments.

    Article Snippet: HSV-1 was boiled in Laemmli buffer containing 200 mM dithiothreitol for 5 min. Proteins were separated by SDS-PAGE on Tris-glycine gels (Thermo Fisher Scientific).

    Techniques: Polyacrylamide Gel Electrophoresis, SDS Page, Western Blot

    HSV-1 gC influences the pH of conformational changes in the fusion protein gB. Extracellular preparations of HSV-1 gCR or ΔgC (∼10 7 genome copies) were treated at pH levels ranging from 7.3 to 5.0 and blotted directly to nitrocellulose. Blots were probed with gB MAbs at neutral pH. Antibody reactivity was quantitated with ImageJ. The reactivity of pH 7.3 samples was set to 100%. Data represent means and standard deviations of results from at least two independent experiments. The pH treatment that reduced reactivity by > 50% is indicated with gray shading (gCR) or red boxes (ΔgC). After each MAb designation, the gB domain (I to VI) containing the MAb epitope is indicated in parentheses (see Fig. S4 in the supplemental material).

    Journal: mSphere

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

    doi: 10.1128/mSphere.00826-19

    Figure Lengend Snippet: HSV-1 gC influences the pH of conformational changes in the fusion protein gB. Extracellular preparations of HSV-1 gCR or ΔgC (∼10 7 genome copies) were treated at pH levels ranging from 7.3 to 5.0 and blotted directly to nitrocellulose. Blots were probed with gB MAbs at neutral pH. Antibody reactivity was quantitated with ImageJ. The reactivity of pH 7.3 samples was set to 100%. Data represent means and standard deviations of results from at least two independent experiments. The pH treatment that reduced reactivity by > 50% is indicated with gray shading (gCR) or red boxes (ΔgC). After each MAb designation, the gB domain (I to VI) containing the MAb epitope is indicated in parentheses (see Fig. S4 in the supplemental material).

    Article Snippet: HSV-1 was boiled in Laemmli buffer containing 200 mM dithiothreitol for 5 min. Proteins were separated by SDS-PAGE on Tris-glycine gels (Thermo Fisher Scientific).

    Techniques:

    HSV-1 lacking gC exhibits reduced entry and infectivity in a subset of cell types. Equivalent inocula of HSV-1 gCR (A) or ΔgC (B) were bound to Vero, CHO-HVEM, IMR-32, or HEKa cells for 1 h at 4 0 C. Following a shift to 37°C for 6 h, infected cells (MOI of ∼0.9) were quantitated by immunofluorescence. Infectivity is reported as percent HSV antigen-positive cells of ∼500 total cells. Data represent means of results from three independent experiments each performed in triplicate, with standard deviations. *, P

    Journal: mSphere

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

    doi: 10.1128/mSphere.00826-19

    Figure Lengend Snippet: HSV-1 lacking gC exhibits reduced entry and infectivity in a subset of cell types. Equivalent inocula of HSV-1 gCR (A) or ΔgC (B) were bound to Vero, CHO-HVEM, IMR-32, or HEKa cells for 1 h at 4 0 C. Following a shift to 37°C for 6 h, infected cells (MOI of ∼0.9) were quantitated by immunofluorescence. Infectivity is reported as percent HSV antigen-positive cells of ∼500 total cells. Data represent means of results from three independent experiments each performed in triplicate, with standard deviations. *, P

    Article Snippet: HSV-1 was boiled in Laemmli buffer containing 200 mM dithiothreitol for 5 min. Proteins were separated by SDS-PAGE on Tris-glycine gels (Thermo Fisher Scientific).

    Techniques: Infection, Immunofluorescence

    HSV-1 ΔgC attachment to cells. (A and B) Approximately 10 6 genome copies of extracellular HSV-1 gCR (A) or ΔgC (B) were added to the indicated prechilled cell monolayers at 4°C for 1 h on ice. Following two PBS washes, cells were trypsinized, and cell-associated HSV-1 was quantitated by qPCR. CHOpgs745 cells lack heparan sulfate receptors for HSV attachment and served as controls. Data represent means of results from three independent experiments performed in quadruplicate with standard errors. *, P

    Journal: mSphere

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

    doi: 10.1128/mSphere.00826-19

    Figure Lengend Snippet: HSV-1 ΔgC attachment to cells. (A and B) Approximately 10 6 genome copies of extracellular HSV-1 gCR (A) or ΔgC (B) were added to the indicated prechilled cell monolayers at 4°C for 1 h on ice. Following two PBS washes, cells were trypsinized, and cell-associated HSV-1 was quantitated by qPCR. CHOpgs745 cells lack heparan sulfate receptors for HSV attachment and served as controls. Data represent means of results from three independent experiments performed in quadruplicate with standard errors. *, P

    Article Snippet: HSV-1 was boiled in Laemmli buffer containing 200 mM dithiothreitol for 5 min. Proteins were separated by SDS-PAGE on Tris-glycine gels (Thermo Fisher Scientific).

    Techniques: Real-time Polymerase Chain Reaction

    Expression of nucleolin (cell-surface target protein) and STAT3 in epithelial cancer cell lines. Immunofluorescence using anti-nucleolin antibody showing nucleolin expression in epithelioid carcinoma A431 ( A ), HNC line FaDu ( B ); ovarian carcinoma OVCAR3 ( C ). Secondary FITC-conjugated IgG (green) was used. Nuclei were counterstained with DAPI (blue). D - nucleolin expression in membrane (M) and nuclear (N) fractions of cells. Lanes 1,2 - A431, Lanes 3,4 - FaDu, lanes 5,6- HeLa cells. E- electrophoretic mobility shift assay (EMSA) showing STAT3d alone (lane 1,3) and after adding FaDu cell lysate (lane 2,4). STAT3 d was labeled either using Cy5.5 (lanes 1,2) or dual-labeled with Cy5.5 and 800CW(lanes3, 4); F- Western blotting of STAT3 in FaDu cell extract (lane 1) and HeLa extract (lane 2, positive control).

    Journal: Nanotheranostics

    Article Title: Dual radiosensitization and anti-STAT3 anti-proliferative strategy based on delivery of gold nanoparticle - oligonucleotide nanoconstructs to head and neck cancer cells.

    doi: 10.7150/ntno.22335

    Figure Lengend Snippet: Expression of nucleolin (cell-surface target protein) and STAT3 in epithelial cancer cell lines. Immunofluorescence using anti-nucleolin antibody showing nucleolin expression in epithelioid carcinoma A431 ( A ), HNC line FaDu ( B ); ovarian carcinoma OVCAR3 ( C ). Secondary FITC-conjugated IgG (green) was used. Nuclei were counterstained with DAPI (blue). D - nucleolin expression in membrane (M) and nuclear (N) fractions of cells. Lanes 1,2 - A431, Lanes 3,4 - FaDu, lanes 5,6- HeLa cells. E- electrophoretic mobility shift assay (EMSA) showing STAT3d alone (lane 1,3) and after adding FaDu cell lysate (lane 2,4). STAT3 d was labeled either using Cy5.5 (lanes 1,2) or dual-labeled with Cy5.5 and 800CW(lanes3, 4); F- Western blotting of STAT3 in FaDu cell extract (lane 1) and HeLa extract (lane 2, positive control).

    Article Snippet: Western blot analysis Heat-denatured DTT-reduced cell protein samples of A431 and FaDu cell lysate isolated by using a subcellular protein fractionation kit (Thermo Fisher Scientific) as well as HeLa whole cell lysate, positive control (Santa Cruz Biotechnology), were resolved by electrophoresis on 4-20% TGX gradient Tris/Tricine/SDS precast gels (Bio-Rad, Hercules CA).

    Techniques: Expressing, Immunofluorescence, Electrophoretic Mobility Shift Assay, Labeling, Western Blot, Positive Control

    The effect of RT on viability (A) of A431 (blue bars) and FaDu cells (red bars) and genomic DNA damage (B) pre- (solid bars) and post- (hatched bars) radiation treatment. Both viability and the damage to DNA were determined by using flow cytometry. Control cells (no pre-incubation), or experimental cells incubated with 1) Cetuximab (control as a standard immunotherapy for head and neck cancer), 2) STAT3d or 3) AuNP-NUAP-STAT3d for 20h and then either mock-irradiated or irradiated with a single dose of 4Gy. Staining performed with FITC- labeled rabbit polyclonal anti- γ-H2AX antibodies and analyzed by FACS. * - Indicates statistical significance, p

    Journal: Nanotheranostics

    Article Title: Dual radiosensitization and anti-STAT3 anti-proliferative strategy based on delivery of gold nanoparticle - oligonucleotide nanoconstructs to head and neck cancer cells.

    doi: 10.7150/ntno.22335

    Figure Lengend Snippet: The effect of RT on viability (A) of A431 (blue bars) and FaDu cells (red bars) and genomic DNA damage (B) pre- (solid bars) and post- (hatched bars) radiation treatment. Both viability and the damage to DNA were determined by using flow cytometry. Control cells (no pre-incubation), or experimental cells incubated with 1) Cetuximab (control as a standard immunotherapy for head and neck cancer), 2) STAT3d or 3) AuNP-NUAP-STAT3d for 20h and then either mock-irradiated or irradiated with a single dose of 4Gy. Staining performed with FITC- labeled rabbit polyclonal anti- γ-H2AX antibodies and analyzed by FACS. * - Indicates statistical significance, p

    Article Snippet: Western blot analysis Heat-denatured DTT-reduced cell protein samples of A431 and FaDu cell lysate isolated by using a subcellular protein fractionation kit (Thermo Fisher Scientific) as well as HeLa whole cell lysate, positive control (Santa Cruz Biotechnology), were resolved by electrophoresis on 4-20% TGX gradient Tris/Tricine/SDS precast gels (Bio-Rad, Hercules CA).

    Techniques: Flow Cytometry, Cytometry, Incubation, Irradiation, Staining, Labeling, FACS

    The uptake of aptamers and aptamer-linked AuNP nanoconstructs in cell culture. A - Total cell uptake of Cy5.5-labeled nucleolin-specific aptamer (NUAP) and control non-specific aptamer (CTAP) incubated with HNC cell line FaDu and squamous carcinoma A431 for 4h. The uptake was determined by using measurements of Cy5.5 fluorescence in cell lysates; B - fractional content of surface-bound and internalized AuNP-aptamer-STAT3d constructs in FaDu and A431 cells. The % of total in the bound and internalized fractions was determined by measuring fluorescence of cell lysates after fractionation (surface and internalized). AuNP constructs were incubated in the cell culture for 4h at 37 o C. C - Internalization of STAT3d, AuNP-STAT3d and AuNP-NUAP-STAT3d in FaDu and A431 cells by [ 99m Tc] radiolabeling of a one of the strand of STAT3d by using modification with MAG3 chelate.

    Journal: Nanotheranostics

    Article Title: Dual radiosensitization and anti-STAT3 anti-proliferative strategy based on delivery of gold nanoparticle - oligonucleotide nanoconstructs to head and neck cancer cells.

    doi: 10.7150/ntno.22335

    Figure Lengend Snippet: The uptake of aptamers and aptamer-linked AuNP nanoconstructs in cell culture. A - Total cell uptake of Cy5.5-labeled nucleolin-specific aptamer (NUAP) and control non-specific aptamer (CTAP) incubated with HNC cell line FaDu and squamous carcinoma A431 for 4h. The uptake was determined by using measurements of Cy5.5 fluorescence in cell lysates; B - fractional content of surface-bound and internalized AuNP-aptamer-STAT3d constructs in FaDu and A431 cells. The % of total in the bound and internalized fractions was determined by measuring fluorescence of cell lysates after fractionation (surface and internalized). AuNP constructs were incubated in the cell culture for 4h at 37 o C. C - Internalization of STAT3d, AuNP-STAT3d and AuNP-NUAP-STAT3d in FaDu and A431 cells by [ 99m Tc] radiolabeling of a one of the strand of STAT3d by using modification with MAG3 chelate.

    Article Snippet: Western blot analysis Heat-denatured DTT-reduced cell protein samples of A431 and FaDu cell lysate isolated by using a subcellular protein fractionation kit (Thermo Fisher Scientific) as well as HeLa whole cell lysate, positive control (Santa Cruz Biotechnology), were resolved by electrophoresis on 4-20% TGX gradient Tris/Tricine/SDS precast gels (Bio-Rad, Hercules CA).

    Techniques: Cell Culture, Labeling, Incubation, Fluorescence, Construct, Fractionation, Radioactivity, Modification

    Confocal multichannel microscopy of STAT3d and AuNP-NUAP-STAT3d uptake in epithelial cancer cells after incubating with A431 cell culture (A, B) or FaDu cells (C, D); AuNP-NUAP-STAT3d (A - A431 cells; C - FaDu cells); STAT3 duplex (B - A431 cells; D - FaDu cells). Blue- DAPI; Green - anti-EGFR-Alexa Fluor488 for staining cell plasma membrane; Red - Alexa Fluor 568 (one strand of STAT3 duplex).

    Journal: Nanotheranostics

    Article Title: Dual radiosensitization and anti-STAT3 anti-proliferative strategy based on delivery of gold nanoparticle - oligonucleotide nanoconstructs to head and neck cancer cells.

    doi: 10.7150/ntno.22335

    Figure Lengend Snippet: Confocal multichannel microscopy of STAT3d and AuNP-NUAP-STAT3d uptake in epithelial cancer cells after incubating with A431 cell culture (A, B) or FaDu cells (C, D); AuNP-NUAP-STAT3d (A - A431 cells; C - FaDu cells); STAT3 duplex (B - A431 cells; D - FaDu cells). Blue- DAPI; Green - anti-EGFR-Alexa Fluor488 for staining cell plasma membrane; Red - Alexa Fluor 568 (one strand of STAT3 duplex).

    Article Snippet: Western blot analysis Heat-denatured DTT-reduced cell protein samples of A431 and FaDu cell lysate isolated by using a subcellular protein fractionation kit (Thermo Fisher Scientific) as well as HeLa whole cell lysate, positive control (Santa Cruz Biotechnology), were resolved by electrophoresis on 4-20% TGX gradient Tris/Tricine/SDS precast gels (Bio-Rad, Hercules CA).

    Techniques: Microscopy, Cell Culture, Staining