Structured Review

DSMZ bhk 21
Reduced MERS-CoV S-driven host cell entry is caused by inefficient S protein binding to DPP4 harboring polymorphic amino acid residues. In order to investigate whether reduced MERS-CoV S-driven host cell entry and MERS-CoV replication is due to inefficient MERS-CoV S binding to DPP4 harboring amino acid polymorphisms at the binding interface, we performed co-immunoprecipitation (co-IP) as well as binding experiments with a soluble S protein comprising the S1 subunit of MERS-CoV S fused to the Fc region of human IgG. (A) 293T cells were cotransfected with expression plasmids coding for soluble, Fc-tagged MERS-CoV S1 (solMERS-S1-Fc) and the indicated DPP4 variant containing a C-terminal cMYC-tag. Cells that were transfected only with empty expression vector alone, or empty expression vector instead of either solMERS-S1-Fc or DPP4 served as controls. At 48 h posttransduction, cells were lysed and incubated with protein A sepharose. Next, samples were subjected to SDS-PAGE and Western blot analysis. DPP4 levels were detected via antibodies specific for the cMYC-tag, whereas solMERS-S1-Fc was detected using a peroxidase-coupled anti-human antibody. Similar results were obtained in three individual experiments. Analysis of whole cell lysates (WCL) for expression of solMERS-S1-Fc, DPP4 and ß-actin confirmed comparable ß-actin levels in each sample and comparable expression levels for solMERS-S1-Fc and DPP4. (B) For quantification of MERS-CoV S/DPP4 interaction we first normalized the DPP4 signals against the respective solMERS-S1-Fc signals. Then, MERS-CoV S/DPP4 interaction was set as 100% for wildtype (WT) DPP4 and the relative interaction efficiency for each DPP4 mutant was calculated accordingly. Presented are the mean data from three independent experiments. Error bars indicate the SEM. Statistical significance of differences in MERS-CoV S/DPP4 interaction between WT and mutant DPP4 was analyzed by one-way analysis of variance with Dunnett’s posttest ( p > 0.05, ns; p ≤ 0.05, *; p ≤ 0.001, ***). (C) Soluble MERS-CoV S1-Fc was incubated with <t>BHK-21</t> cells expressing wildtype (WT) or mutant DPP4, or cells transfected with empty expression vector or an ACE2-expression plasmid (controls). To detect bound S protein, the cells were subsequently incubated with an AlexaFluor488-conjugated anti-human antibody directed against the Fc-tag. Fluorescent signals representing bound solMERS-S1-Fc were analyzed by flow cytometry and MFI values for each sample were calculated. For normalization, the MFI value of the negative control (empty expression vector) was subtracted from all samples. Further, binding of solMERS-S1-Fc to cells expressing DPP4 WT was set as 100% and the relative binding to cells expressing the DPP4 mutants was calculated accordingly. Shown are the combined data of five independent experiments with error bars indicating the SEM. Statistical significance of differences in solMERS-S1-Fc binding to cells expressing WT or mutant DPP4 was analyzed by one-way analysis of variance with Dunnett’s posttest ( p > 0.05, ns; p ≤ 0.05, *; p ≤ 0.01, **; p ≤ 0.001, ***).
Bhk 21, supplied by DSMZ, used in various techniques. Bioz Stars score: 93/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/bhk 21/product/DSMZ
Average 93 stars, based on 12 article reviews
Price from $9.99 to $1999.99
bhk 21 - by Bioz Stars, 2020-09
93/100 stars

Images

1) Product Images from "Polymorphisms in dipeptidyl peptidase 4 reduce host cell entry of Middle East respiratory syndrome coronavirus"

Article Title: Polymorphisms in dipeptidyl peptidase 4 reduce host cell entry of Middle East respiratory syndrome coronavirus

Journal: Emerging Microbes & Infections

doi: 10.1080/22221751.2020.1713705

Reduced MERS-CoV S-driven host cell entry is caused by inefficient S protein binding to DPP4 harboring polymorphic amino acid residues. In order to investigate whether reduced MERS-CoV S-driven host cell entry and MERS-CoV replication is due to inefficient MERS-CoV S binding to DPP4 harboring amino acid polymorphisms at the binding interface, we performed co-immunoprecipitation (co-IP) as well as binding experiments with a soluble S protein comprising the S1 subunit of MERS-CoV S fused to the Fc region of human IgG. (A) 293T cells were cotransfected with expression plasmids coding for soluble, Fc-tagged MERS-CoV S1 (solMERS-S1-Fc) and the indicated DPP4 variant containing a C-terminal cMYC-tag. Cells that were transfected only with empty expression vector alone, or empty expression vector instead of either solMERS-S1-Fc or DPP4 served as controls. At 48 h posttransduction, cells were lysed and incubated with protein A sepharose. Next, samples were subjected to SDS-PAGE and Western blot analysis. DPP4 levels were detected via antibodies specific for the cMYC-tag, whereas solMERS-S1-Fc was detected using a peroxidase-coupled anti-human antibody. Similar results were obtained in three individual experiments. Analysis of whole cell lysates (WCL) for expression of solMERS-S1-Fc, DPP4 and ß-actin confirmed comparable ß-actin levels in each sample and comparable expression levels for solMERS-S1-Fc and DPP4. (B) For quantification of MERS-CoV S/DPP4 interaction we first normalized the DPP4 signals against the respective solMERS-S1-Fc signals. Then, MERS-CoV S/DPP4 interaction was set as 100% for wildtype (WT) DPP4 and the relative interaction efficiency for each DPP4 mutant was calculated accordingly. Presented are the mean data from three independent experiments. Error bars indicate the SEM. Statistical significance of differences in MERS-CoV S/DPP4 interaction between WT and mutant DPP4 was analyzed by one-way analysis of variance with Dunnett’s posttest ( p > 0.05, ns; p ≤ 0.05, *; p ≤ 0.001, ***). (C) Soluble MERS-CoV S1-Fc was incubated with BHK-21 cells expressing wildtype (WT) or mutant DPP4, or cells transfected with empty expression vector or an ACE2-expression plasmid (controls). To detect bound S protein, the cells were subsequently incubated with an AlexaFluor488-conjugated anti-human antibody directed against the Fc-tag. Fluorescent signals representing bound solMERS-S1-Fc were analyzed by flow cytometry and MFI values for each sample were calculated. For normalization, the MFI value of the negative control (empty expression vector) was subtracted from all samples. Further, binding of solMERS-S1-Fc to cells expressing DPP4 WT was set as 100% and the relative binding to cells expressing the DPP4 mutants was calculated accordingly. Shown are the combined data of five independent experiments with error bars indicating the SEM. Statistical significance of differences in solMERS-S1-Fc binding to cells expressing WT or mutant DPP4 was analyzed by one-way analysis of variance with Dunnett’s posttest ( p > 0.05, ns; p ≤ 0.05, *; p ≤ 0.01, **; p ≤ 0.001, ***).
Figure Legend Snippet: Reduced MERS-CoV S-driven host cell entry is caused by inefficient S protein binding to DPP4 harboring polymorphic amino acid residues. In order to investigate whether reduced MERS-CoV S-driven host cell entry and MERS-CoV replication is due to inefficient MERS-CoV S binding to DPP4 harboring amino acid polymorphisms at the binding interface, we performed co-immunoprecipitation (co-IP) as well as binding experiments with a soluble S protein comprising the S1 subunit of MERS-CoV S fused to the Fc region of human IgG. (A) 293T cells were cotransfected with expression plasmids coding for soluble, Fc-tagged MERS-CoV S1 (solMERS-S1-Fc) and the indicated DPP4 variant containing a C-terminal cMYC-tag. Cells that were transfected only with empty expression vector alone, or empty expression vector instead of either solMERS-S1-Fc or DPP4 served as controls. At 48 h posttransduction, cells were lysed and incubated with protein A sepharose. Next, samples were subjected to SDS-PAGE and Western blot analysis. DPP4 levels were detected via antibodies specific for the cMYC-tag, whereas solMERS-S1-Fc was detected using a peroxidase-coupled anti-human antibody. Similar results were obtained in three individual experiments. Analysis of whole cell lysates (WCL) for expression of solMERS-S1-Fc, DPP4 and ß-actin confirmed comparable ß-actin levels in each sample and comparable expression levels for solMERS-S1-Fc and DPP4. (B) For quantification of MERS-CoV S/DPP4 interaction we first normalized the DPP4 signals against the respective solMERS-S1-Fc signals. Then, MERS-CoV S/DPP4 interaction was set as 100% for wildtype (WT) DPP4 and the relative interaction efficiency for each DPP4 mutant was calculated accordingly. Presented are the mean data from three independent experiments. Error bars indicate the SEM. Statistical significance of differences in MERS-CoV S/DPP4 interaction between WT and mutant DPP4 was analyzed by one-way analysis of variance with Dunnett’s posttest ( p > 0.05, ns; p ≤ 0.05, *; p ≤ 0.001, ***). (C) Soluble MERS-CoV S1-Fc was incubated with BHK-21 cells expressing wildtype (WT) or mutant DPP4, or cells transfected with empty expression vector or an ACE2-expression plasmid (controls). To detect bound S protein, the cells were subsequently incubated with an AlexaFluor488-conjugated anti-human antibody directed against the Fc-tag. Fluorescent signals representing bound solMERS-S1-Fc were analyzed by flow cytometry and MFI values for each sample were calculated. For normalization, the MFI value of the negative control (empty expression vector) was subtracted from all samples. Further, binding of solMERS-S1-Fc to cells expressing DPP4 WT was set as 100% and the relative binding to cells expressing the DPP4 mutants was calculated accordingly. Shown are the combined data of five independent experiments with error bars indicating the SEM. Statistical significance of differences in solMERS-S1-Fc binding to cells expressing WT or mutant DPP4 was analyzed by one-way analysis of variance with Dunnett’s posttest ( p > 0.05, ns; p ≤ 0.05, *; p ≤ 0.01, **; p ≤ 0.001, ***).

Techniques Used: Protein Binding, Binding Assay, Immunoprecipitation, Co-Immunoprecipitation Assay, Expressing, Variant Assay, Transfection, Plasmid Preparation, Incubation, SDS Page, Western Blot, Mutagenesis, Flow Cytometry, Negative Control

Identification of polymorphic amino acid residues in DPP4 that do not support efficient MERS-CoV S-driven host cell entry. (A) To investigate whether mutant DPP4 support host cell entry driven by MERS-CoV S, we produced vesicular stomatitis virus pseudotype particles (VSVpp) harboring MERS-CoV S (left) or VSV G (control, right). VSVpp were further inoculated on BHK-21 cells expressing wildtype (WT) or mutant DPP4, or cells that were transfected with empty expression vector. At 16 h posttransduction, transduction efficiency was analyzed by measuring the activity of virus-encoded firefly luciferase. Shown are the combined data from three independent experiments (each performed in quadruplicates) for which transduction efficiency of cells expressing DPP4 WT was set as 100%. Error bars indicate the SEM. Statistical significance of differences in transduction efficiency of cells expressing WT or mutant DPP4 was analyzed by one-way analysis of variance with Dunnett’s posttest ( p > 0.05, not significant [ns]; p ≤ 0.05, *; p ≤ 0.01, **; p ≤ 0.001, ***).
Figure Legend Snippet: Identification of polymorphic amino acid residues in DPP4 that do not support efficient MERS-CoV S-driven host cell entry. (A) To investigate whether mutant DPP4 support host cell entry driven by MERS-CoV S, we produced vesicular stomatitis virus pseudotype particles (VSVpp) harboring MERS-CoV S (left) or VSV G (control, right). VSVpp were further inoculated on BHK-21 cells expressing wildtype (WT) or mutant DPP4, or cells that were transfected with empty expression vector. At 16 h posttransduction, transduction efficiency was analyzed by measuring the activity of virus-encoded firefly luciferase. Shown are the combined data from three independent experiments (each performed in quadruplicates) for which transduction efficiency of cells expressing DPP4 WT was set as 100%. Error bars indicate the SEM. Statistical significance of differences in transduction efficiency of cells expressing WT or mutant DPP4 was analyzed by one-way analysis of variance with Dunnett’s posttest ( p > 0.05, not significant [ns]; p ≤ 0.05, *; p ≤ 0.01, **; p ≤ 0.001, ***).

Techniques Used: Mutagenesis, Produced, Expressing, Transfection, Plasmid Preparation, Transduction, Activity Assay, Luciferase

DPP4 harboring polymorphic amino acid residues at the binding interface with MERS-CoV S are efficiently transported to the cell surface. (A) Wildtype (WT) and mutant DPP4 were expressed in BHK-21 cells (cells transfected with empty expression vector served as negative control). Surface expressed DPP4 was stained by subsequent incubation of the non-permeabilized cells with a primary antibody that targets the DPP4 ectodomain and an AlexaFluor488-conjugated secondary antibody. Fluorescent signals representing surface-expressed DPP4 were analyzed by flow cytometry and the mean fluorescence intensity (MFI) values for each sample were calculated. For normalization, the MFI value of the negative control was subtracted from all samples. Further, surface expression of DPP4 WT was set as 100% and the relative surface expression of the DPP4 mutants was calculated accordingly. Shown are the combined data of three experiments with error bars indicating the SEM. Statistical significance for differences in surface expression between WT and mutant DPP4 was tested by one-way analysis of variance with Dunnett’s posttest ( p > 0.05, not significant; p ≤ 0.05, *). (B) DPP4 surface expression was further analyzed by immunofluorescence analysis. For this, DPP4 WT or DPP4 mutants were expressed in BHK-21 cells grown on coverslips (cells transfected with empty expression vector served as negative control). After fixation of the cells, surface expressed DPP4 was stained by subsequent incubation of non-permeabilized cells with a primary antibody that targets the DPP4 ectodomain and an AlexaFluor568-conjugated secondary antibody. In addition, cellular nuclei were stained with DAPI. Finally, images were taken using a confocal laser scanning microscope at a magnification of 80x.
Figure Legend Snippet: DPP4 harboring polymorphic amino acid residues at the binding interface with MERS-CoV S are efficiently transported to the cell surface. (A) Wildtype (WT) and mutant DPP4 were expressed in BHK-21 cells (cells transfected with empty expression vector served as negative control). Surface expressed DPP4 was stained by subsequent incubation of the non-permeabilized cells with a primary antibody that targets the DPP4 ectodomain and an AlexaFluor488-conjugated secondary antibody. Fluorescent signals representing surface-expressed DPP4 were analyzed by flow cytometry and the mean fluorescence intensity (MFI) values for each sample were calculated. For normalization, the MFI value of the negative control was subtracted from all samples. Further, surface expression of DPP4 WT was set as 100% and the relative surface expression of the DPP4 mutants was calculated accordingly. Shown are the combined data of three experiments with error bars indicating the SEM. Statistical significance for differences in surface expression between WT and mutant DPP4 was tested by one-way analysis of variance with Dunnett’s posttest ( p > 0.05, not significant; p ≤ 0.05, *). (B) DPP4 surface expression was further analyzed by immunofluorescence analysis. For this, DPP4 WT or DPP4 mutants were expressed in BHK-21 cells grown on coverslips (cells transfected with empty expression vector served as negative control). After fixation of the cells, surface expressed DPP4 was stained by subsequent incubation of non-permeabilized cells with a primary antibody that targets the DPP4 ectodomain and an AlexaFluor568-conjugated secondary antibody. In addition, cellular nuclei were stained with DAPI. Finally, images were taken using a confocal laser scanning microscope at a magnification of 80x.

Techniques Used: Binding Assay, Mutagenesis, Transfection, Expressing, Plasmid Preparation, Negative Control, Staining, Incubation, Flow Cytometry, Fluorescence, Immunofluorescence, Laser-Scanning Microscopy

DPP4 harboring polymorphic amino acid residues at the binding interface with MERS-CoV S poorly support replication of live MERS-CoV. Two DPP4 mutants that showed reduced compatibility for MERS-CoV S-driven host cell entry (K267E and A291P) were analyzed in the context of infection and replication of authentic MERS-CoV. For this, BHK-21 cells expressing wildtype (WT) or mutant DPP4, or no DPP4 at all (negative control) were inoculated with MERS-CoV. At 1 h postinfection, the inoculum was removed and the cells were washed before they received fresh culture medium and were further incubated. MERS-CoV replication was analyzed at 0, 24 and 48 h postinfection by determining MERS-CoV genome equivalents (GE) in the culture supernatant (given as GE/ml) by quantitative reverse-transcriptase PCR. Shown are the combined results of three independent experiments (each performed in triplicates). Error bars indicate the SEM. Statistical significance of differences in MERS-CoV replication in cells expressing WT or mutant DPP4 was analyzed by two-way analysis of variance with Dunnett’s posttest ( p > 0.05, ns; p ≤ 0.05, *).
Figure Legend Snippet: DPP4 harboring polymorphic amino acid residues at the binding interface with MERS-CoV S poorly support replication of live MERS-CoV. Two DPP4 mutants that showed reduced compatibility for MERS-CoV S-driven host cell entry (K267E and A291P) were analyzed in the context of infection and replication of authentic MERS-CoV. For this, BHK-21 cells expressing wildtype (WT) or mutant DPP4, or no DPP4 at all (negative control) were inoculated with MERS-CoV. At 1 h postinfection, the inoculum was removed and the cells were washed before they received fresh culture medium and were further incubated. MERS-CoV replication was analyzed at 0, 24 and 48 h postinfection by determining MERS-CoV genome equivalents (GE) in the culture supernatant (given as GE/ml) by quantitative reverse-transcriptase PCR. Shown are the combined results of three independent experiments (each performed in triplicates). Error bars indicate the SEM. Statistical significance of differences in MERS-CoV replication in cells expressing WT or mutant DPP4 was analyzed by two-way analysis of variance with Dunnett’s posttest ( p > 0.05, ns; p ≤ 0.05, *).

Techniques Used: Binding Assay, Infection, Expressing, Mutagenesis, Negative Control, Incubation, Polymerase Chain Reaction

2) Product Images from "An Fc Double-Engineered CD20 Antibody with Enhanced Ability to Trigger Complement-Dependent Cytotoxicity and Antibody-Dependent Cell-Mediated Cytotoxicity"

Article Title: An Fc Double-Engineered CD20 Antibody with Enhanced Ability to Trigger Complement-Dependent Cytotoxicity and Antibody-Dependent Cell-Mediated Cytotoxicity

Journal: Transfusion Medicine and Hemotherapy

doi: 10.1159/000479978

Induction of CDC by rituximab antibody variants. A Daudi cells were coated with RTX-wt-CHO, RTX-EFTAE-CHO, RTX-wt-Lec13 or RTX-EFTAE-Lec13 (concentration: 50 µg/ml) and then incubated in the presence of human serum (1%) as a source of C1q. Eculizumab was added to block CDC. Deposition of C1q was analyzed with a FITC-coupled mouse anti-human C1q antibody by flow cytometry. Bars represent mean values ± SEM (n = 3). B CDC by rituximab variants in comparison to corresponding HER2-specific control antibodies was analyzed by 51 Cr release experiments using Daudi cells as targets in the presence of 25% human plasma. Antibodies were applied at 10 µg/ml. Mean values ± SEM are depicted. Significant differences between CD20 antibodies and similarly designed control proteins are indicated (*, p ≤ 0.05; n = 3). C Dose-dependent induction of CDC against Daudi (n = 3), GRANTA-519 (n = 4) and MEC-2 (n = 4) cells by rituximab variants. Human plasma (25%) was added as a source of complement. Statistically significant differences in CDC between engineered antibodies and the native CD20 IgG1 molecule are indicated (*, P ≤ 0.05). D Daudi, GRANTA-519 and MEC-2 cells were incubated with specific antibodies against mCRPs CD46, CD55 or CD59 (blue peaks) or isotype matched control antibodies (white peaks), which were subsequently detected with secondary FITC-conjugated goat anti-mouse IgG Fc F(ab') 2 fragments, and expression levels were analyzed by flow cytometry. Results from one representative experiment are shown (n = 3; MFI, mean fluorescence intensity).
Figure Legend Snippet: Induction of CDC by rituximab antibody variants. A Daudi cells were coated with RTX-wt-CHO, RTX-EFTAE-CHO, RTX-wt-Lec13 or RTX-EFTAE-Lec13 (concentration: 50 µg/ml) and then incubated in the presence of human serum (1%) as a source of C1q. Eculizumab was added to block CDC. Deposition of C1q was analyzed with a FITC-coupled mouse anti-human C1q antibody by flow cytometry. Bars represent mean values ± SEM (n = 3). B CDC by rituximab variants in comparison to corresponding HER2-specific control antibodies was analyzed by 51 Cr release experiments using Daudi cells as targets in the presence of 25% human plasma. Antibodies were applied at 10 µg/ml. Mean values ± SEM are depicted. Significant differences between CD20 antibodies and similarly designed control proteins are indicated (*, p ≤ 0.05; n = 3). C Dose-dependent induction of CDC against Daudi (n = 3), GRANTA-519 (n = 4) and MEC-2 (n = 4) cells by rituximab variants. Human plasma (25%) was added as a source of complement. Statistically significant differences in CDC between engineered antibodies and the native CD20 IgG1 molecule are indicated (*, P ≤ 0.05). D Daudi, GRANTA-519 and MEC-2 cells were incubated with specific antibodies against mCRPs CD46, CD55 or CD59 (blue peaks) or isotype matched control antibodies (white peaks), which were subsequently detected with secondary FITC-conjugated goat anti-mouse IgG Fc F(ab') 2 fragments, and expression levels were analyzed by flow cytometry. Results from one representative experiment are shown (n = 3; MFI, mean fluorescence intensity).

Techniques Used: Concentration Assay, Incubation, Blocking Assay, Flow Cytometry, Cytometry, Expressing, Fluorescence

CD20 binding analysis. A CD20-positive MEC-2 cells were incubated in buffer alone (white peaks) or in the presence of the indicated antibodies at 50 µg/ml (green peaks), then reacted with FITC-conjugated anti-human IgG Fc F(ab') 2 and analyzed by flow cytometry. B RTX-wt-CHO, RTX-EFTAE-CHO, RTX-wt-Lec13 and RTX-EFTAE-Lec13 (concentration: 50 µg/ml) specifically bound to CHO-K1-CD20 cells but did not react with non-transfected CHO-K1 cells. Bars indicate mean values ± SEM (n = 2). Antibodies were detected with FITC-conjugated anti-human IgG Fc F(ab') 2 fragments and flow cytometry. Trastuzumab was used as control antibody (MFI, mean fluorescence intensity). C Antibody variants were analyzed for binding to CHO-K1-CD20 cells at varying concentrations using secondary FITC-conjugated anti-human IgG Fc F(ab') 2 fragments for detection and flow cytometry. Data points represent mean values ± SEM (n = 4).
Figure Legend Snippet: CD20 binding analysis. A CD20-positive MEC-2 cells were incubated in buffer alone (white peaks) or in the presence of the indicated antibodies at 50 µg/ml (green peaks), then reacted with FITC-conjugated anti-human IgG Fc F(ab') 2 and analyzed by flow cytometry. B RTX-wt-CHO, RTX-EFTAE-CHO, RTX-wt-Lec13 and RTX-EFTAE-Lec13 (concentration: 50 µg/ml) specifically bound to CHO-K1-CD20 cells but did not react with non-transfected CHO-K1 cells. Bars indicate mean values ± SEM (n = 2). Antibodies were detected with FITC-conjugated anti-human IgG Fc F(ab') 2 fragments and flow cytometry. Trastuzumab was used as control antibody (MFI, mean fluorescence intensity). C Antibody variants were analyzed for binding to CHO-K1-CD20 cells at varying concentrations using secondary FITC-conjugated anti-human IgG Fc F(ab') 2 fragments for detection and flow cytometry. Data points represent mean values ± SEM (n = 4).

Techniques Used: Binding Assay, Incubation, Flow Cytometry, Cytometry, Concentration Assay, Transfection, Fluorescence

FcγRIIIA binding and induction of ADCC by rituximab antibody variants. A Dose-dependent binding to BHK cells transfected with expression vectors encoding FcεRI γ chain and either human FcγRIIIA-158V (BHK-CD16-158V) or FcγRIIIA-158V (BHK-CD16-158F) was analyzed by flow cytometry using FITC-coupled anti-human IgG Fc F(ab') 2 fragments. B Antigen-specific binding was verified by analyzing binding to BHK-CD16-158V vs. un-transfected BHK cells. Antibodies were applied at 50 µg/ml and detected as described above. C ADCC by rituximab variants in comparison to corresponding HER2-specific control antibodies was analyzed by 51 Cr release experiments with Daudi cells as targets and human MNCs as effector cells. Antibodies were applied at 10 µg/ml. Mean values ± SEM are depicted. Significant differences between CD20 antibodies and similarly designed control antibodies are indicated (*, p ≤ 0.05; n = 3). D Dose-dependent induction of ADCC against Daudi (n = 3), MEC-2 (n = 4) and GRANTA-519 cells (n = 4) by rituximab variants using MNC effector cells. Statistically significant differences in target cell lysis between Fc-engineered antibodies and the native CD20 IgG1 molecule are indicated (*, p ≤ 0.05).
Figure Legend Snippet: FcγRIIIA binding and induction of ADCC by rituximab antibody variants. A Dose-dependent binding to BHK cells transfected with expression vectors encoding FcεRI γ chain and either human FcγRIIIA-158V (BHK-CD16-158V) or FcγRIIIA-158V (BHK-CD16-158F) was analyzed by flow cytometry using FITC-coupled anti-human IgG Fc F(ab') 2 fragments. B Antigen-specific binding was verified by analyzing binding to BHK-CD16-158V vs. un-transfected BHK cells. Antibodies were applied at 50 µg/ml and detected as described above. C ADCC by rituximab variants in comparison to corresponding HER2-specific control antibodies was analyzed by 51 Cr release experiments with Daudi cells as targets and human MNCs as effector cells. Antibodies were applied at 10 µg/ml. Mean values ± SEM are depicted. Significant differences between CD20 antibodies and similarly designed control antibodies are indicated (*, p ≤ 0.05; n = 3). D Dose-dependent induction of ADCC against Daudi (n = 3), MEC-2 (n = 4) and GRANTA-519 cells (n = 4) by rituximab variants using MNC effector cells. Statistically significant differences in target cell lysis between Fc-engineered antibodies and the native CD20 IgG1 molecule are indicated (*, p ≤ 0.05).

Techniques Used: Binding Assay, Transfection, Expressing, Flow Cytometry, Cytometry, Lysis

3) Product Images from "Nonreplicative RNA Recombination of an Animal Plus-Strand RNA Virus in the Absence of Efficient Translation of Viral Proteins"

Article Title: Nonreplicative RNA Recombination of an Animal Plus-Strand RNA Virus in the Absence of Efficient Translation of Viral Proteins

Journal: Genome Biology and Evolution

doi: 10.1093/gbe/evx046

Processing of fusion proteins of recombinants R-1, R-2, and R-3. ( A ) Schematic representation of authentic (P-1, P-2, P-3) and mutated (P-2P, P-3P) fusion proteins transiently expressed in the MVA-T7 system. P-1 encompasses the wt proteins N pro and C, P-2 and P-3 comprise N pro and the C fusion proteins encoded by the recombinant viruses R-2 and R-3, respectively. In P-2P and P-3P, the asterisks mark the serine to proline substitutions directly downstream of the ubiquitin fragments. Black arrows indicate autoproteolytic cleavage mediated by N pro . White arrows (P-2 and P-3) indicate partial processing directly downstream of the ubiquitin fragments. ( B and C ) Western blot analysis. BHK-21 cells were infected with MVA-T7 and transfected with pCITE-N pro -C constructs encoding P-1, P-2, P-2P, P-3, and P-3P, respectively. Nontransfected cells (−) served as negative control. Cells were lysed 20 h posttransfection and analyzed by Western blot using MAb 1F7 (panel B ) and MAb 13B6 (panel C ) to detect C (C-ubi-C) and N pro , respectively.
Figure Legend Snippet: Processing of fusion proteins of recombinants R-1, R-2, and R-3. ( A ) Schematic representation of authentic (P-1, P-2, P-3) and mutated (P-2P, P-3P) fusion proteins transiently expressed in the MVA-T7 system. P-1 encompasses the wt proteins N pro and C, P-2 and P-3 comprise N pro and the C fusion proteins encoded by the recombinant viruses R-2 and R-3, respectively. In P-2P and P-3P, the asterisks mark the serine to proline substitutions directly downstream of the ubiquitin fragments. Black arrows indicate autoproteolytic cleavage mediated by N pro . White arrows (P-2 and P-3) indicate partial processing directly downstream of the ubiquitin fragments. ( B and C ) Western blot analysis. BHK-21 cells were infected with MVA-T7 and transfected with pCITE-N pro -C constructs encoding P-1, P-2, P-2P, P-3, and P-3P, respectively. Nontransfected cells (−) served as negative control. Cells were lysed 20 h posttransfection and analyzed by Western blot using MAb 1F7 (panel B ) and MAb 13B6 (panel C ) to detect C (C-ubi-C) and N pro , respectively.

Techniques Used: Recombinant, Western Blot, Infection, Transfection, Construct, Negative Control

Related Articles

Polymerase Chain Reaction:

Article Title: Intramolecular domain dynamics regulate synaptic MAGUK protein interactions
Article Snippet: .. HEK-293T (DSMZ no. ACC 635) cell identity was confirmed by fluorescent nonaplex PCR of short tandem repeat markers by DSMZ. .. COS-7 (DSMZ no. ACC 60) cell identity was confirmed by Hinf I-(gtg)5 DNA profiling by DSMZ.

Modification:

Article Title: Polymorphisms in dipeptidyl peptidase 4 reduce host cell entry of Middle East respiratory syndrome coronavirus
Article Snippet: .. Cell culture 293T (human kidney cells, DSMZ no. ACC 635), BHK-21 (hamster kidney cells, DSMZ no. ACC 61) and Vero 76 (African green monkey kidney cells, kindly provided by Andrea Maisner, Philipps-University Marburg) were cultivated in Dulbecco’s modified Eagle medium (PAN-Biotech) while Caco-2 cells (human colorectal adenocarcinoma cells) were cultivated in Minimum Essential Medium (ThermoFisher Scientific). .. The media were supplemented with 10% fetal bovine serum (Biochrom), 100 U/ml of penicillin and 0.1 mg/ml of streptomycin (PAN-Biotech).

Viability Assay:

Article Title: STAT3 mediates C6-ceramide-induced cell death in chronic lymphocytic leukemia
Article Snippet: .. Cell viability assay A set of experiments were conducted to determine the toxicity of CNL and Stattic in JVM-3 cells, Mec-2 cells, CLL patient cells and in normal donor PBMCs. .. Cell viability was assessed by a CellTiter 96 Aqueous One Solution assay kit (Promega, Madison, WI, USA) following the manufacturer’s instructions.

Cell Culture:

Article Title: A hexanucleotide selected for increased cellular uptake in cis contains a highly active CpG-motif in human B cells and primary peripheral blood mononuclear cells
Article Snippet: .. As a cellular model system we chose BHK cells, which were shown earlier to be capable of taking up naked DNA spontaneously and which serve as an established cell culture model. More importantly, there was a relatively low level of uptake of naked ON in BHK cells whereas human B-lymphoid cell lines including BJA-B cells showed high levels of uptake (data not shown). .. Since the combinatorial approach used here does not involve amplification of selected sequences a single-round selection has to be performed.

Article Title: STAT3 mediates C6-ceramide-induced cell death in chronic lymphocytic leukemia
Article Snippet: .. Mec-2 cells (DSMZ), a CLL cell line with mutated p53 were cultured in Iscove’s MDM media supplemented with 10% FBS. .. HEK-293FT cells (Invitrogen) were cultured in D-MEM supplemented with 10% FBS and 1× anti-anti antibiotic (Gibco, Waltham, MA) containing 10 k units ml−1 of penicillin, 10 k μg ml−1 of streptomycin and 25 μg ml−1 of Gibco Amphotericin.

Article Title: Polymorphisms in dipeptidyl peptidase 4 reduce host cell entry of Middle East respiratory syndrome coronavirus
Article Snippet: .. Cell culture 293T (human kidney cells, DSMZ no. ACC 635), BHK-21 (hamster kidney cells, DSMZ no. ACC 61) and Vero 76 (African green monkey kidney cells, kindly provided by Andrea Maisner, Philipps-University Marburg) were cultivated in Dulbecco’s modified Eagle medium (PAN-Biotech) while Caco-2 cells (human colorectal adenocarcinoma cells) were cultivated in Minimum Essential Medium (ThermoFisher Scientific). .. The media were supplemented with 10% fetal bovine serum (Biochrom), 100 U/ml of penicillin and 0.1 mg/ml of streptomycin (PAN-Biotech).

Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • bhk 21  (DSMZ)
    93
    DSMZ bhk 21
    Reduced MERS-CoV S-driven host cell entry is caused by inefficient S protein binding to DPP4 harboring polymorphic amino acid residues. In order to investigate whether reduced MERS-CoV S-driven host cell entry and MERS-CoV replication is due to inefficient MERS-CoV S binding to DPP4 harboring amino acid polymorphisms at the binding interface, we performed co-immunoprecipitation (co-IP) as well as binding experiments with a soluble S protein comprising the S1 subunit of MERS-CoV S fused to the Fc region of human IgG. (A) 293T cells were cotransfected with expression plasmids coding for soluble, Fc-tagged MERS-CoV S1 (solMERS-S1-Fc) and the indicated DPP4 variant containing a C-terminal cMYC-tag. Cells that were transfected only with empty expression vector alone, or empty expression vector instead of either solMERS-S1-Fc or DPP4 served as controls. At 48 h posttransduction, cells were lysed and incubated with protein A sepharose. Next, samples were subjected to SDS-PAGE and Western blot analysis. DPP4 levels were detected via antibodies specific for the cMYC-tag, whereas solMERS-S1-Fc was detected using a peroxidase-coupled anti-human antibody. Similar results were obtained in three individual experiments. Analysis of whole cell lysates (WCL) for expression of solMERS-S1-Fc, DPP4 and ß-actin confirmed comparable ß-actin levels in each sample and comparable expression levels for solMERS-S1-Fc and DPP4. (B) For quantification of MERS-CoV S/DPP4 interaction we first normalized the DPP4 signals against the respective solMERS-S1-Fc signals. Then, MERS-CoV S/DPP4 interaction was set as 100% for wildtype (WT) DPP4 and the relative interaction efficiency for each DPP4 mutant was calculated accordingly. Presented are the mean data from three independent experiments. Error bars indicate the SEM. Statistical significance of differences in MERS-CoV S/DPP4 interaction between WT and mutant DPP4 was analyzed by one-way analysis of variance with Dunnett’s posttest ( p > 0.05, ns; p ≤ 0.05, *; p ≤ 0.001, ***). (C) Soluble MERS-CoV S1-Fc was incubated with <t>BHK-21</t> cells expressing wildtype (WT) or mutant DPP4, or cells transfected with empty expression vector or an ACE2-expression plasmid (controls). To detect bound S protein, the cells were subsequently incubated with an AlexaFluor488-conjugated anti-human antibody directed against the Fc-tag. Fluorescent signals representing bound solMERS-S1-Fc were analyzed by flow cytometry and MFI values for each sample were calculated. For normalization, the MFI value of the negative control (empty expression vector) was subtracted from all samples. Further, binding of solMERS-S1-Fc to cells expressing DPP4 WT was set as 100% and the relative binding to cells expressing the DPP4 mutants was calculated accordingly. Shown are the combined data of five independent experiments with error bars indicating the SEM. Statistical significance of differences in solMERS-S1-Fc binding to cells expressing WT or mutant DPP4 was analyzed by one-way analysis of variance with Dunnett’s posttest ( p > 0.05, ns; p ≤ 0.05, *; p ≤ 0.01, **; p ≤ 0.001, ***).
    Bhk 21, supplied by DSMZ, used in various techniques. Bioz Stars score: 93/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bhk 21/product/DSMZ
    Average 93 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    bhk 21 - by Bioz Stars, 2020-09
    93/100 stars
      Buy from Supplier

    91
    DSMZ baby hamster kidney bhk 21 cells
    Processing of fusion proteins of recombinants R-1, R-2, and R-3. ( A ) Schematic representation of authentic (P-1, P-2, P-3) and mutated (P-2P, P-3P) fusion proteins transiently expressed in the MVA-T7 system. P-1 encompasses the wt proteins N pro and C, P-2 and P-3 comprise N pro and the C fusion proteins encoded by the recombinant viruses R-2 and R-3, respectively. In P-2P and P-3P, the asterisks mark the serine to proline substitutions directly downstream of the ubiquitin fragments. Black arrows indicate autoproteolytic cleavage mediated by N pro . White arrows (P-2 and P-3) indicate partial processing directly downstream of the ubiquitin fragments. ( B and C ) Western blot analysis. <t>BHK-21</t> cells were infected with MVA-T7 and transfected with pCITE-N pro -C constructs encoding P-1, P-2, P-2P, P-3, and P-3P, respectively. Nontransfected cells (−) served as negative control. Cells were lysed 20 h posttransfection and analyzed by Western blot using MAb 1F7 (panel B ) and MAb 13B6 (panel C ) to detect C (C-ubi-C) and N pro , respectively.
    Baby Hamster Kidney Bhk 21 Cells, supplied by DSMZ, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/baby hamster kidney bhk 21 cells/product/DSMZ
    Average 91 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    baby hamster kidney bhk 21 cells - by Bioz Stars, 2020-09
    91/100 stars
      Buy from Supplier

    92
    DSMZ bhk cells
    Processing of fusion proteins of recombinants R-1, R-2, and R-3. ( A ) Schematic representation of authentic (P-1, P-2, P-3) and mutated (P-2P, P-3P) fusion proteins transiently expressed in the MVA-T7 system. P-1 encompasses the wt proteins N pro and C, P-2 and P-3 comprise N pro and the C fusion proteins encoded by the recombinant viruses R-2 and R-3, respectively. In P-2P and P-3P, the asterisks mark the serine to proline substitutions directly downstream of the ubiquitin fragments. Black arrows indicate autoproteolytic cleavage mediated by N pro . White arrows (P-2 and P-3) indicate partial processing directly downstream of the ubiquitin fragments. ( B and C ) Western blot analysis. <t>BHK-21</t> cells were infected with MVA-T7 and transfected with pCITE-N pro -C constructs encoding P-1, P-2, P-2P, P-3, and P-3P, respectively. Nontransfected cells (−) served as negative control. Cells were lysed 20 h posttransfection and analyzed by Western blot using MAb 1F7 (panel B ) and MAb 13B6 (panel C ) to detect C (C-ubi-C) and N pro , respectively.
    Bhk Cells, supplied by DSMZ, used in various techniques. Bioz Stars score: 92/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bhk cells/product/DSMZ
    Average 92 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    bhk cells - by Bioz Stars, 2020-09
    92/100 stars
      Buy from Supplier

    Image Search Results


    Reduced MERS-CoV S-driven host cell entry is caused by inefficient S protein binding to DPP4 harboring polymorphic amino acid residues. In order to investigate whether reduced MERS-CoV S-driven host cell entry and MERS-CoV replication is due to inefficient MERS-CoV S binding to DPP4 harboring amino acid polymorphisms at the binding interface, we performed co-immunoprecipitation (co-IP) as well as binding experiments with a soluble S protein comprising the S1 subunit of MERS-CoV S fused to the Fc region of human IgG. (A) 293T cells were cotransfected with expression plasmids coding for soluble, Fc-tagged MERS-CoV S1 (solMERS-S1-Fc) and the indicated DPP4 variant containing a C-terminal cMYC-tag. Cells that were transfected only with empty expression vector alone, or empty expression vector instead of either solMERS-S1-Fc or DPP4 served as controls. At 48 h posttransduction, cells were lysed and incubated with protein A sepharose. Next, samples were subjected to SDS-PAGE and Western blot analysis. DPP4 levels were detected via antibodies specific for the cMYC-tag, whereas solMERS-S1-Fc was detected using a peroxidase-coupled anti-human antibody. Similar results were obtained in three individual experiments. Analysis of whole cell lysates (WCL) for expression of solMERS-S1-Fc, DPP4 and ß-actin confirmed comparable ß-actin levels in each sample and comparable expression levels for solMERS-S1-Fc and DPP4. (B) For quantification of MERS-CoV S/DPP4 interaction we first normalized the DPP4 signals against the respective solMERS-S1-Fc signals. Then, MERS-CoV S/DPP4 interaction was set as 100% for wildtype (WT) DPP4 and the relative interaction efficiency for each DPP4 mutant was calculated accordingly. Presented are the mean data from three independent experiments. Error bars indicate the SEM. Statistical significance of differences in MERS-CoV S/DPP4 interaction between WT and mutant DPP4 was analyzed by one-way analysis of variance with Dunnett’s posttest ( p > 0.05, ns; p ≤ 0.05, *; p ≤ 0.001, ***). (C) Soluble MERS-CoV S1-Fc was incubated with BHK-21 cells expressing wildtype (WT) or mutant DPP4, or cells transfected with empty expression vector or an ACE2-expression plasmid (controls). To detect bound S protein, the cells were subsequently incubated with an AlexaFluor488-conjugated anti-human antibody directed against the Fc-tag. Fluorescent signals representing bound solMERS-S1-Fc were analyzed by flow cytometry and MFI values for each sample were calculated. For normalization, the MFI value of the negative control (empty expression vector) was subtracted from all samples. Further, binding of solMERS-S1-Fc to cells expressing DPP4 WT was set as 100% and the relative binding to cells expressing the DPP4 mutants was calculated accordingly. Shown are the combined data of five independent experiments with error bars indicating the SEM. Statistical significance of differences in solMERS-S1-Fc binding to cells expressing WT or mutant DPP4 was analyzed by one-way analysis of variance with Dunnett’s posttest ( p > 0.05, ns; p ≤ 0.05, *; p ≤ 0.01, **; p ≤ 0.001, ***).

    Journal: Emerging Microbes & Infections

    Article Title: Polymorphisms in dipeptidyl peptidase 4 reduce host cell entry of Middle East respiratory syndrome coronavirus

    doi: 10.1080/22221751.2020.1713705

    Figure Lengend Snippet: Reduced MERS-CoV S-driven host cell entry is caused by inefficient S protein binding to DPP4 harboring polymorphic amino acid residues. In order to investigate whether reduced MERS-CoV S-driven host cell entry and MERS-CoV replication is due to inefficient MERS-CoV S binding to DPP4 harboring amino acid polymorphisms at the binding interface, we performed co-immunoprecipitation (co-IP) as well as binding experiments with a soluble S protein comprising the S1 subunit of MERS-CoV S fused to the Fc region of human IgG. (A) 293T cells were cotransfected with expression plasmids coding for soluble, Fc-tagged MERS-CoV S1 (solMERS-S1-Fc) and the indicated DPP4 variant containing a C-terminal cMYC-tag. Cells that were transfected only with empty expression vector alone, or empty expression vector instead of either solMERS-S1-Fc or DPP4 served as controls. At 48 h posttransduction, cells were lysed and incubated with protein A sepharose. Next, samples were subjected to SDS-PAGE and Western blot analysis. DPP4 levels were detected via antibodies specific for the cMYC-tag, whereas solMERS-S1-Fc was detected using a peroxidase-coupled anti-human antibody. Similar results were obtained in three individual experiments. Analysis of whole cell lysates (WCL) for expression of solMERS-S1-Fc, DPP4 and ß-actin confirmed comparable ß-actin levels in each sample and comparable expression levels for solMERS-S1-Fc and DPP4. (B) For quantification of MERS-CoV S/DPP4 interaction we first normalized the DPP4 signals against the respective solMERS-S1-Fc signals. Then, MERS-CoV S/DPP4 interaction was set as 100% for wildtype (WT) DPP4 and the relative interaction efficiency for each DPP4 mutant was calculated accordingly. Presented are the mean data from three independent experiments. Error bars indicate the SEM. Statistical significance of differences in MERS-CoV S/DPP4 interaction between WT and mutant DPP4 was analyzed by one-way analysis of variance with Dunnett’s posttest ( p > 0.05, ns; p ≤ 0.05, *; p ≤ 0.001, ***). (C) Soluble MERS-CoV S1-Fc was incubated with BHK-21 cells expressing wildtype (WT) or mutant DPP4, or cells transfected with empty expression vector or an ACE2-expression plasmid (controls). To detect bound S protein, the cells were subsequently incubated with an AlexaFluor488-conjugated anti-human antibody directed against the Fc-tag. Fluorescent signals representing bound solMERS-S1-Fc were analyzed by flow cytometry and MFI values for each sample were calculated. For normalization, the MFI value of the negative control (empty expression vector) was subtracted from all samples. Further, binding of solMERS-S1-Fc to cells expressing DPP4 WT was set as 100% and the relative binding to cells expressing the DPP4 mutants was calculated accordingly. Shown are the combined data of five independent experiments with error bars indicating the SEM. Statistical significance of differences in solMERS-S1-Fc binding to cells expressing WT or mutant DPP4 was analyzed by one-way analysis of variance with Dunnett’s posttest ( p > 0.05, ns; p ≤ 0.05, *; p ≤ 0.01, **; p ≤ 0.001, ***).

    Article Snippet: Cell culture 293T (human kidney cells, DSMZ no. ACC 635), BHK-21 (hamster kidney cells, DSMZ no. ACC 61) and Vero 76 (African green monkey kidney cells, kindly provided by Andrea Maisner, Philipps-University Marburg) were cultivated in Dulbecco’s modified Eagle medium (PAN-Biotech) while Caco-2 cells (human colorectal adenocarcinoma cells) were cultivated in Minimum Essential Medium (ThermoFisher Scientific).

    Techniques: Protein Binding, Binding Assay, Immunoprecipitation, Co-Immunoprecipitation Assay, Expressing, Variant Assay, Transfection, Plasmid Preparation, Incubation, SDS Page, Western Blot, Mutagenesis, Flow Cytometry, Negative Control

    Identification of polymorphic amino acid residues in DPP4 that do not support efficient MERS-CoV S-driven host cell entry. (A) To investigate whether mutant DPP4 support host cell entry driven by MERS-CoV S, we produced vesicular stomatitis virus pseudotype particles (VSVpp) harboring MERS-CoV S (left) or VSV G (control, right). VSVpp were further inoculated on BHK-21 cells expressing wildtype (WT) or mutant DPP4, or cells that were transfected with empty expression vector. At 16 h posttransduction, transduction efficiency was analyzed by measuring the activity of virus-encoded firefly luciferase. Shown are the combined data from three independent experiments (each performed in quadruplicates) for which transduction efficiency of cells expressing DPP4 WT was set as 100%. Error bars indicate the SEM. Statistical significance of differences in transduction efficiency of cells expressing WT or mutant DPP4 was analyzed by one-way analysis of variance with Dunnett’s posttest ( p > 0.05, not significant [ns]; p ≤ 0.05, *; p ≤ 0.01, **; p ≤ 0.001, ***).

    Journal: Emerging Microbes & Infections

    Article Title: Polymorphisms in dipeptidyl peptidase 4 reduce host cell entry of Middle East respiratory syndrome coronavirus

    doi: 10.1080/22221751.2020.1713705

    Figure Lengend Snippet: Identification of polymorphic amino acid residues in DPP4 that do not support efficient MERS-CoV S-driven host cell entry. (A) To investigate whether mutant DPP4 support host cell entry driven by MERS-CoV S, we produced vesicular stomatitis virus pseudotype particles (VSVpp) harboring MERS-CoV S (left) or VSV G (control, right). VSVpp were further inoculated on BHK-21 cells expressing wildtype (WT) or mutant DPP4, or cells that were transfected with empty expression vector. At 16 h posttransduction, transduction efficiency was analyzed by measuring the activity of virus-encoded firefly luciferase. Shown are the combined data from three independent experiments (each performed in quadruplicates) for which transduction efficiency of cells expressing DPP4 WT was set as 100%. Error bars indicate the SEM. Statistical significance of differences in transduction efficiency of cells expressing WT or mutant DPP4 was analyzed by one-way analysis of variance with Dunnett’s posttest ( p > 0.05, not significant [ns]; p ≤ 0.05, *; p ≤ 0.01, **; p ≤ 0.001, ***).

    Article Snippet: Cell culture 293T (human kidney cells, DSMZ no. ACC 635), BHK-21 (hamster kidney cells, DSMZ no. ACC 61) and Vero 76 (African green monkey kidney cells, kindly provided by Andrea Maisner, Philipps-University Marburg) were cultivated in Dulbecco’s modified Eagle medium (PAN-Biotech) while Caco-2 cells (human colorectal adenocarcinoma cells) were cultivated in Minimum Essential Medium (ThermoFisher Scientific).

    Techniques: Mutagenesis, Produced, Expressing, Transfection, Plasmid Preparation, Transduction, Activity Assay, Luciferase

    DPP4 harboring polymorphic amino acid residues at the binding interface with MERS-CoV S are efficiently transported to the cell surface. (A) Wildtype (WT) and mutant DPP4 were expressed in BHK-21 cells (cells transfected with empty expression vector served as negative control). Surface expressed DPP4 was stained by subsequent incubation of the non-permeabilized cells with a primary antibody that targets the DPP4 ectodomain and an AlexaFluor488-conjugated secondary antibody. Fluorescent signals representing surface-expressed DPP4 were analyzed by flow cytometry and the mean fluorescence intensity (MFI) values for each sample were calculated. For normalization, the MFI value of the negative control was subtracted from all samples. Further, surface expression of DPP4 WT was set as 100% and the relative surface expression of the DPP4 mutants was calculated accordingly. Shown are the combined data of three experiments with error bars indicating the SEM. Statistical significance for differences in surface expression between WT and mutant DPP4 was tested by one-way analysis of variance with Dunnett’s posttest ( p > 0.05, not significant; p ≤ 0.05, *). (B) DPP4 surface expression was further analyzed by immunofluorescence analysis. For this, DPP4 WT or DPP4 mutants were expressed in BHK-21 cells grown on coverslips (cells transfected with empty expression vector served as negative control). After fixation of the cells, surface expressed DPP4 was stained by subsequent incubation of non-permeabilized cells with a primary antibody that targets the DPP4 ectodomain and an AlexaFluor568-conjugated secondary antibody. In addition, cellular nuclei were stained with DAPI. Finally, images were taken using a confocal laser scanning microscope at a magnification of 80x.

    Journal: Emerging Microbes & Infections

    Article Title: Polymorphisms in dipeptidyl peptidase 4 reduce host cell entry of Middle East respiratory syndrome coronavirus

    doi: 10.1080/22221751.2020.1713705

    Figure Lengend Snippet: DPP4 harboring polymorphic amino acid residues at the binding interface with MERS-CoV S are efficiently transported to the cell surface. (A) Wildtype (WT) and mutant DPP4 were expressed in BHK-21 cells (cells transfected with empty expression vector served as negative control). Surface expressed DPP4 was stained by subsequent incubation of the non-permeabilized cells with a primary antibody that targets the DPP4 ectodomain and an AlexaFluor488-conjugated secondary antibody. Fluorescent signals representing surface-expressed DPP4 were analyzed by flow cytometry and the mean fluorescence intensity (MFI) values for each sample were calculated. For normalization, the MFI value of the negative control was subtracted from all samples. Further, surface expression of DPP4 WT was set as 100% and the relative surface expression of the DPP4 mutants was calculated accordingly. Shown are the combined data of three experiments with error bars indicating the SEM. Statistical significance for differences in surface expression between WT and mutant DPP4 was tested by one-way analysis of variance with Dunnett’s posttest ( p > 0.05, not significant; p ≤ 0.05, *). (B) DPP4 surface expression was further analyzed by immunofluorescence analysis. For this, DPP4 WT or DPP4 mutants were expressed in BHK-21 cells grown on coverslips (cells transfected with empty expression vector served as negative control). After fixation of the cells, surface expressed DPP4 was stained by subsequent incubation of non-permeabilized cells with a primary antibody that targets the DPP4 ectodomain and an AlexaFluor568-conjugated secondary antibody. In addition, cellular nuclei were stained with DAPI. Finally, images were taken using a confocal laser scanning microscope at a magnification of 80x.

    Article Snippet: Cell culture 293T (human kidney cells, DSMZ no. ACC 635), BHK-21 (hamster kidney cells, DSMZ no. ACC 61) and Vero 76 (African green monkey kidney cells, kindly provided by Andrea Maisner, Philipps-University Marburg) were cultivated in Dulbecco’s modified Eagle medium (PAN-Biotech) while Caco-2 cells (human colorectal adenocarcinoma cells) were cultivated in Minimum Essential Medium (ThermoFisher Scientific).

    Techniques: Binding Assay, Mutagenesis, Transfection, Expressing, Plasmid Preparation, Negative Control, Staining, Incubation, Flow Cytometry, Fluorescence, Immunofluorescence, Laser-Scanning Microscopy

    DPP4 harboring polymorphic amino acid residues at the binding interface with MERS-CoV S poorly support replication of live MERS-CoV. Two DPP4 mutants that showed reduced compatibility for MERS-CoV S-driven host cell entry (K267E and A291P) were analyzed in the context of infection and replication of authentic MERS-CoV. For this, BHK-21 cells expressing wildtype (WT) or mutant DPP4, or no DPP4 at all (negative control) were inoculated with MERS-CoV. At 1 h postinfection, the inoculum was removed and the cells were washed before they received fresh culture medium and were further incubated. MERS-CoV replication was analyzed at 0, 24 and 48 h postinfection by determining MERS-CoV genome equivalents (GE) in the culture supernatant (given as GE/ml) by quantitative reverse-transcriptase PCR. Shown are the combined results of three independent experiments (each performed in triplicates). Error bars indicate the SEM. Statistical significance of differences in MERS-CoV replication in cells expressing WT or mutant DPP4 was analyzed by two-way analysis of variance with Dunnett’s posttest ( p > 0.05, ns; p ≤ 0.05, *).

    Journal: Emerging Microbes & Infections

    Article Title: Polymorphisms in dipeptidyl peptidase 4 reduce host cell entry of Middle East respiratory syndrome coronavirus

    doi: 10.1080/22221751.2020.1713705

    Figure Lengend Snippet: DPP4 harboring polymorphic amino acid residues at the binding interface with MERS-CoV S poorly support replication of live MERS-CoV. Two DPP4 mutants that showed reduced compatibility for MERS-CoV S-driven host cell entry (K267E and A291P) were analyzed in the context of infection and replication of authentic MERS-CoV. For this, BHK-21 cells expressing wildtype (WT) or mutant DPP4, or no DPP4 at all (negative control) were inoculated with MERS-CoV. At 1 h postinfection, the inoculum was removed and the cells were washed before they received fresh culture medium and were further incubated. MERS-CoV replication was analyzed at 0, 24 and 48 h postinfection by determining MERS-CoV genome equivalents (GE) in the culture supernatant (given as GE/ml) by quantitative reverse-transcriptase PCR. Shown are the combined results of three independent experiments (each performed in triplicates). Error bars indicate the SEM. Statistical significance of differences in MERS-CoV replication in cells expressing WT or mutant DPP4 was analyzed by two-way analysis of variance with Dunnett’s posttest ( p > 0.05, ns; p ≤ 0.05, *).

    Article Snippet: Cell culture 293T (human kidney cells, DSMZ no. ACC 635), BHK-21 (hamster kidney cells, DSMZ no. ACC 61) and Vero 76 (African green monkey kidney cells, kindly provided by Andrea Maisner, Philipps-University Marburg) were cultivated in Dulbecco’s modified Eagle medium (PAN-Biotech) while Caco-2 cells (human colorectal adenocarcinoma cells) were cultivated in Minimum Essential Medium (ThermoFisher Scientific).

    Techniques: Binding Assay, Infection, Expressing, Mutagenesis, Negative Control, Incubation, Polymerase Chain Reaction

    Processing of fusion proteins of recombinants R-1, R-2, and R-3. ( A ) Schematic representation of authentic (P-1, P-2, P-3) and mutated (P-2P, P-3P) fusion proteins transiently expressed in the MVA-T7 system. P-1 encompasses the wt proteins N pro and C, P-2 and P-3 comprise N pro and the C fusion proteins encoded by the recombinant viruses R-2 and R-3, respectively. In P-2P and P-3P, the asterisks mark the serine to proline substitutions directly downstream of the ubiquitin fragments. Black arrows indicate autoproteolytic cleavage mediated by N pro . White arrows (P-2 and P-3) indicate partial processing directly downstream of the ubiquitin fragments. ( B and C ) Western blot analysis. BHK-21 cells were infected with MVA-T7 and transfected with pCITE-N pro -C constructs encoding P-1, P-2, P-2P, P-3, and P-3P, respectively. Nontransfected cells (−) served as negative control. Cells were lysed 20 h posttransfection and analyzed by Western blot using MAb 1F7 (panel B ) and MAb 13B6 (panel C ) to detect C (C-ubi-C) and N pro , respectively.

    Journal: Genome Biology and Evolution

    Article Title: Nonreplicative RNA Recombination of an Animal Plus-Strand RNA Virus in the Absence of Efficient Translation of Viral Proteins

    doi: 10.1093/gbe/evx046

    Figure Lengend Snippet: Processing of fusion proteins of recombinants R-1, R-2, and R-3. ( A ) Schematic representation of authentic (P-1, P-2, P-3) and mutated (P-2P, P-3P) fusion proteins transiently expressed in the MVA-T7 system. P-1 encompasses the wt proteins N pro and C, P-2 and P-3 comprise N pro and the C fusion proteins encoded by the recombinant viruses R-2 and R-3, respectively. In P-2P and P-3P, the asterisks mark the serine to proline substitutions directly downstream of the ubiquitin fragments. Black arrows indicate autoproteolytic cleavage mediated by N pro . White arrows (P-2 and P-3) indicate partial processing directly downstream of the ubiquitin fragments. ( B and C ) Western blot analysis. BHK-21 cells were infected with MVA-T7 and transfected with pCITE-N pro -C constructs encoding P-1, P-2, P-2P, P-3, and P-3P, respectively. Nontransfected cells (−) served as negative control. Cells were lysed 20 h posttransfection and analyzed by Western blot using MAb 1F7 (panel B ) and MAb 13B6 (panel C ) to detect C (C-ubi-C) and N pro , respectively.

    Article Snippet: Baby hamster kidney (BHK-21) cells were obtained from the DSMZ (Braunschweig, Germany) and maintained in EDulb medium supplemented with 5% fetal calf serum.

    Techniques: Recombinant, Western Blot, Infection, Transfection, Construct, Negative Control