gst tagged myo1e sh3 domain  (New England Biolabs)


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    Name:
    Factor Xa Protease
    Description:
    Factor Xa Protease 250 ug
    Catalog Number:
    p8010l
    Price:
    305
    Size:
    250 ug
    Category:
    Proteases
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    Structured Review

    New England Biolabs gst tagged myo1e sh3 domain
    Factor Xa Protease
    Factor Xa Protease 250 ug
    https://www.bioz.com/result/gst tagged myo1e sh3 domain/product/New England Biolabs
    Average 90 stars, based on 113 article reviews
    Price from $9.99 to $1999.99
    gst tagged myo1e sh3 domain - by Bioz Stars, 2021-02
    90/100 stars

    Images

    1) Product Images from "Myosin-1E interacts with FAK proline-rich region 1 to induce fibronectin-type matrix"

    Article Title: Myosin-1E interacts with FAK proline-rich region 1 to induce fibronectin-type matrix

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1614894114

    MYO1E activates FAK to induce SPP1 expression in fibroblasts. ( A ) In vitro FAK kinase activity of full-length recombinant FAK versus FAK kinase domain only (aa 393–698) in the presence or absence of 1:2 titrated recombinant MYO1E SH3 (mean ±
    Figure Legend Snippet: MYO1E activates FAK to induce SPP1 expression in fibroblasts. ( A ) In vitro FAK kinase activity of full-length recombinant FAK versus FAK kinase domain only (aa 393–698) in the presence or absence of 1:2 titrated recombinant MYO1E SH3 (mean ±

    Techniques Used: Expressing, In Vitro, Activity Assay, Recombinant

    Assessment of folding of recombinant MYO1E SH3 domains by circular dichroism and NMR. ( A ) Circular dichroism spectra of recombinant WT and W1088K MYO1E SH3 domains (after GST-tag removal). Results indicate folded proteins. ( B and C ) Assignment of the
    Figure Legend Snippet: Assessment of folding of recombinant MYO1E SH3 domains by circular dichroism and NMR. ( A ) Circular dichroism spectra of recombinant WT and W1088K MYO1E SH3 domains (after GST-tag removal). Results indicate folded proteins. ( B and C ) Assignment of the

    Techniques Used: Recombinant, Nuclear Magnetic Resonance

    Analytical SEC demonstrates complex formation between the recombinant MYO1E-SH3 domain (after GST-tag removal) and RALPSIK peptide in solution.
    Figure Legend Snippet: Analytical SEC demonstrates complex formation between the recombinant MYO1E-SH3 domain (after GST-tag removal) and RALPSIK peptide in solution.

    Techniques Used: Size-exclusion Chromatography, Recombinant

    2) Product Images from "Recombinant Measles Viruses Efficiently Entering Cells through Targeted Receptors"

    Article Title: Recombinant Measles Viruses Efficiently Entering Cells through Targeted Receptors

    Journal: Journal of Virology

    doi:

    Genomic structure, protein composition, and replication of recombinant MV. (A) Plasmid p(+)MV-NSe coding for the MV antigenome (top), Pac I- Spe I fragments used for subcloning (center), and amino acid sequences (one-letter code) of the junctions between the H protein ectodomain and the specificity domains (bottom). Coding regions of the six MV cistrons are represented by solid black boxes, the transmembrane segment of the F and H proteins by a horizontally lined box, the FXa cleavage site and the flexible linker by a hatched box, and the ligand by a gray box. Arginine residues in parentheses have been deleted. For details see text. (B) Protein composition of recombinant viruses. Viral particles were purified by centrifugation through a 20% sucrose layer onto a 60% sucrose cushion and subsequent pelleting. The viruses were titrated, 5,000 PFU was subjected to lysis, and proteins were separated by SDS-PAGE. For immunodetection, a MV-specific antiserum was used. (C) FXa protease sensitivity of hybrid H proteins. Purified viral particles (5,000 PFU) were lysed and incubated for 1 h without (−) or with (+) FXa protease at room temperature. Proteins were separated by SDS-PAGE and detected with an H-specific antiserum. (D) FACS analysis of CD46 (interrupted line), IGF1r (dotted line), and EGFr (continuous line) expression on Vero cells. Vertical axis, cell number; horizontal axis, fluorescence intensity. The greyed profile represents incubation of the cells without the primary antibody. (E) Time course of released (left panel) and cell-associated (right panel) virus production in Vero cells infected with parental MV (diamonds), MV-H/XhEGF (squares), and MV-H/XhIGF1 (triangles). Cells were infected at a MOI of 3 and were incubated at 32°C for the times indicated. Viral titers were determined by 50% end-point dilution. Indicated values are averages of three experiments.
    Figure Legend Snippet: Genomic structure, protein composition, and replication of recombinant MV. (A) Plasmid p(+)MV-NSe coding for the MV antigenome (top), Pac I- Spe I fragments used for subcloning (center), and amino acid sequences (one-letter code) of the junctions between the H protein ectodomain and the specificity domains (bottom). Coding regions of the six MV cistrons are represented by solid black boxes, the transmembrane segment of the F and H proteins by a horizontally lined box, the FXa cleavage site and the flexible linker by a hatched box, and the ligand by a gray box. Arginine residues in parentheses have been deleted. For details see text. (B) Protein composition of recombinant viruses. Viral particles were purified by centrifugation through a 20% sucrose layer onto a 60% sucrose cushion and subsequent pelleting. The viruses were titrated, 5,000 PFU was subjected to lysis, and proteins were separated by SDS-PAGE. For immunodetection, a MV-specific antiserum was used. (C) FXa protease sensitivity of hybrid H proteins. Purified viral particles (5,000 PFU) were lysed and incubated for 1 h without (−) or with (+) FXa protease at room temperature. Proteins were separated by SDS-PAGE and detected with an H-specific antiserum. (D) FACS analysis of CD46 (interrupted line), IGF1r (dotted line), and EGFr (continuous line) expression on Vero cells. Vertical axis, cell number; horizontal axis, fluorescence intensity. The greyed profile represents incubation of the cells without the primary antibody. (E) Time course of released (left panel) and cell-associated (right panel) virus production in Vero cells infected with parental MV (diamonds), MV-H/XhEGF (squares), and MV-H/XhIGF1 (triangles). Cells were infected at a MOI of 3 and were incubated at 32°C for the times indicated. Viral titers were determined by 50% end-point dilution. Indicated values are averages of three experiments.

    Techniques Used: Recombinant, Plasmid Preparation, Subcloning, Purification, Centrifugation, Lysis, SDS Page, Immunodetection, Incubation, FACS, Expressing, Fluorescence, Infection

    Competition of viral entry by soluble receptors (A) and the effect of proteolytic cleavage of the specificity domain on entry (B). (A) Competition of viral infections by soluble EGF (sEGF; top panel) or soluble IGF1 (sIGF1; bottom panel). CHO-hEGFr and Vero cells pretreated or not with sEGF were infected with MV green or MV green -H/XhEGF at a MOI of 0.3. 3T3-hIGF1r and Vero cells pretreated or not with sIGF1 were infected with MV green and MV green -H/XhIGF1 at a MOI of 0.1. The number of infected cells was determined at 24 h p.i. by counting GFP-expressing cells in a standard area. Percentages are relative to the number of GFP-expressing cells in infections without added soluble receptor. Results obtained with MV Edmonston on Vero cells are indicated by gray columns. Results obtained with recombinant MV on Vero cells are indicated by white columns and on rodent cells by black columns. (B) Effects of the treatment of recombinant viruses with FXa protease prior to infection. Vero and CHO-hEGFr or Vero and 3T3-hIGF1r cells were infected with MV green -H/XhEGF or MV green -H/XhIGF1 (10 4 PFU/well), respectively, and were pretreated with 0, 5, or 50 μg of FXa protease/ml. The number of GFP-expressing cells was determined 42 h p.i. Percentages refer to the number of cells infected with untreated virus. For details, see text.
    Figure Legend Snippet: Competition of viral entry by soluble receptors (A) and the effect of proteolytic cleavage of the specificity domain on entry (B). (A) Competition of viral infections by soluble EGF (sEGF; top panel) or soluble IGF1 (sIGF1; bottom panel). CHO-hEGFr and Vero cells pretreated or not with sEGF were infected with MV green or MV green -H/XhEGF at a MOI of 0.3. 3T3-hIGF1r and Vero cells pretreated or not with sIGF1 were infected with MV green and MV green -H/XhIGF1 at a MOI of 0.1. The number of infected cells was determined at 24 h p.i. by counting GFP-expressing cells in a standard area. Percentages are relative to the number of GFP-expressing cells in infections without added soluble receptor. Results obtained with MV Edmonston on Vero cells are indicated by gray columns. Results obtained with recombinant MV on Vero cells are indicated by white columns and on rodent cells by black columns. (B) Effects of the treatment of recombinant viruses with FXa protease prior to infection. Vero and CHO-hEGFr or Vero and 3T3-hIGF1r cells were infected with MV green -H/XhEGF or MV green -H/XhIGF1 (10 4 PFU/well), respectively, and were pretreated with 0, 5, or 50 μg of FXa protease/ml. The number of GFP-expressing cells was determined 42 h p.i. Percentages refer to the number of cells infected with untreated virus. For details, see text.

    Techniques Used: Infection, Expressing, Recombinant

    3) Product Images from "Using Cystine Knot Proteins as a Novel Approach to Retarget Oncolytic Measles Virus"

    Article Title: Using Cystine Knot Proteins as a Novel Approach to Retarget Oncolytic Measles Virus

    Journal: Molecular Therapy Oncolytics

    doi: 10.1016/j.omto.2017.09.005

    Proteolytic Removal of the CKP Prevents Infection by MV-CKPint Virus MV-CKPint was treated with FXa protease at 100 and 200 μg/mL for 2 hr. Untreated and protease-treated virus was used to infect human glioblastoma (U87), melanoma (MDAMB-435) and vero cells for 3 hr at 37°C. The number of live cells was quantified 96 hr post-infection using the trypan blue exclusion method (bar graphs). The experiments were performed in three independent repeats. Error bars represent SEM. *p
    Figure Legend Snippet: Proteolytic Removal of the CKP Prevents Infection by MV-CKPint Virus MV-CKPint was treated with FXa protease at 100 and 200 μg/mL for 2 hr. Untreated and protease-treated virus was used to infect human glioblastoma (U87), melanoma (MDAMB-435) and vero cells for 3 hr at 37°C. The number of live cells was quantified 96 hr post-infection using the trypan blue exclusion method (bar graphs). The experiments were performed in three independent repeats. Error bars represent SEM. *p

    Techniques Used: Infection

    4) Product Images from ""

    Article Title:

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M110.193490

    Schematic diagram of the topology of Gwt1p. A , summary of the results of glycosylation ( Glyco ) mapping and fXa cleavage analysis. Four conserved regions ( A–D ) and essential residues (Asp-145, Lys-155, Asp-165, and Arg-216) are also shown. Asp-145
    Figure Legend Snippet: Schematic diagram of the topology of Gwt1p. A , summary of the results of glycosylation ( Glyco ) mapping and fXa cleavage analysis. Four conserved regions ( A–D ) and essential residues (Asp-145, Lys-155, Asp-165, and Arg-216) are also shown. Asp-145

    Techniques Used:

    Determination of membrane topology of Gwt1p by factor Xa protease cleavage analysis. Membrane fractions from GC1 cells expressing wild-type ( WT ) or Gwt1p fusion proteins with fXa cleavage sequences, tagged with an HA epitope, were prepared and digested
    Figure Legend Snippet: Determination of membrane topology of Gwt1p by factor Xa protease cleavage analysis. Membrane fractions from GC1 cells expressing wild-type ( WT ) or Gwt1p fusion proteins with fXa cleavage sequences, tagged with an HA epitope, were prepared and digested

    Techniques Used: Expressing

    Related Articles

    Infection:

    Article Title: Recombinant Measles Viruses Efficiently Entering Cells through Targeted Receptors
    Article Snippet: .. Analogously, proteolytic cleavage of MV-H/XhIGF1 virus particles with FXa protease resulted in the loss of more than 80% of the infectivity selectively in 3T3-hIGF1r cells (Fig. B, right panel, compare black and white columns). .. Thus, by two different approaches, competition with a soluble form of the displayed domain and proteolytic cleavage of that domain from viral particles, it was confirmed that entry of recombinant MV in rodent cells depends on the interaction of the specificity domain with the cognate receptor.

    Article Title: Using Cystine Knot Proteins as a Novel Approach to Retarget Oncolytic Measles Virus
    Article Snippet: .. For the FXa proteolysis assay, prior to cellular infection, the number of MV-CKPint virus equivalent to an MOI of 1 was incubated with FXa protease (New England Biolabs, Ipswich, MA, USA) at final concentrations of 100 and 200 μg/mL in Opti-MEM media. .. The virus was incubated with FXa at room temperature for 1 hr and then for an additional 1 hr at 37°C in the water bath.

    Purification:

    Article Title: Functional Alteration of a Dimeric Insecticidal Lectin to a Monomeric Antifungal Protein Correlated to Its Oligomeric Status
    Article Snippet: .. Cleavage of the fusion protein The fusion protein was digested with Factor Xa (New England Biolabs, Ma USA) at 25°C using the pMAL protein fusion and purification system kit (New England Biolab, Ma USA) according to the manufacturer's protocol. ..

    Incubation:

    Article Title: Fab-based bispecific antibody formats with robust biophysical properties and biological activity
    Article Snippet: .. Factor Xa (1 μL per 50 μg of IgG-Fab protein – New England BioLabs) was added and incubated at 4°C overnight to cleave the Fab from the IgG. .. Factor Xa was removed by adding 100 μl of Xarrest agarose (EMD Millipore) per 8 μL Factor Xa and incubated at room temperature for 1 hr.

    Article Title: Using Cystine Knot Proteins as a Novel Approach to Retarget Oncolytic Measles Virus
    Article Snippet: .. For the FXa proteolysis assay, prior to cellular infection, the number of MV-CKPint virus equivalent to an MOI of 1 was incubated with FXa protease (New England Biolabs, Ipswich, MA, USA) at final concentrations of 100 and 200 μg/mL in Opti-MEM media. .. The virus was incubated with FXa at room temperature for 1 hr and then for an additional 1 hr at 37°C in the water bath.

    other:

    Article Title:
    Article Snippet: When fXa cleavage sites were inserted into loop 5–6 ( 6 ), loop 7–8 ( 9 ), or loop 9–10 ( 12 ) of Gwt1p (see also A ), the fXa protease was accessible to the fusion proteins in the presence or absence of digitonin, suggesting that these positions were located on the cytosolic side of the ER membrane.

    Article Title: Substrate Control in Stereoselective Lanthionine Biosynthesis
    Article Snippet: Trypsin was purchased from Worthington Biochemical Corporation; Factor Xa was obtained from New England Biolabs and other endoproteinases were ordered from Roche Biosciences.

    Staining:

    Article Title: Barley Metallothioneins: MT3 and MT4 Are Localized in the Grain Aleurone Layer and Show Differential Zinc Binding 1Barley Metallothioneins: MT3 and MT4 Are Localized in the Grain Aleurone Layer and Show Differential Zinc Binding 1 [W]Barley Metallothioneins: MT3 and MT4 Are Localized in the Grain Aleurone Layer and Show Differential Zinc Binding 1 [W] [OA]
    Article Snippet: .. Recombinant proteins were documented by SDS-PAGE using Tris-Tricine 8% gels and stained with PageBlue protein stain (Fermentas). was cleaved from MBP using Factor Xa as recommended (New England Biolabs) and concentrated using YM30 and YM3 microcon centrifugal filter devices (Millipore) with 30- and 3-kD cutoffs, respectively. ..

    Recombinant:

    Article Title: Barley Metallothioneins: MT3 and MT4 Are Localized in the Grain Aleurone Layer and Show Differential Zinc Binding 1Barley Metallothioneins: MT3 and MT4 Are Localized in the Grain Aleurone Layer and Show Differential Zinc Binding 1 [W]Barley Metallothioneins: MT3 and MT4 Are Localized in the Grain Aleurone Layer and Show Differential Zinc Binding 1 [W] [OA]
    Article Snippet: .. Recombinant proteins were documented by SDS-PAGE using Tris-Tricine 8% gels and stained with PageBlue protein stain (Fermentas). was cleaved from MBP using Factor Xa as recommended (New England Biolabs) and concentrated using YM30 and YM3 microcon centrifugal filter devices (Millipore) with 30- and 3-kD cutoffs, respectively. ..

    Proteolysis Assay:

    Article Title: Using Cystine Knot Proteins as a Novel Approach to Retarget Oncolytic Measles Virus
    Article Snippet: .. For the FXa proteolysis assay, prior to cellular infection, the number of MV-CKPint virus equivalent to an MOI of 1 was incubated with FXa protease (New England Biolabs, Ipswich, MA, USA) at final concentrations of 100 and 200 μg/mL in Opti-MEM media. .. The virus was incubated with FXa at room temperature for 1 hr and then for an additional 1 hr at 37°C in the water bath.

    SDS Page:

    Article Title: Barley Metallothioneins: MT3 and MT4 Are Localized in the Grain Aleurone Layer and Show Differential Zinc Binding 1Barley Metallothioneins: MT3 and MT4 Are Localized in the Grain Aleurone Layer and Show Differential Zinc Binding 1 [W]Barley Metallothioneins: MT3 and MT4 Are Localized in the Grain Aleurone Layer and Show Differential Zinc Binding 1 [W] [OA]
    Article Snippet: .. Recombinant proteins were documented by SDS-PAGE using Tris-Tricine 8% gels and stained with PageBlue protein stain (Fermentas). was cleaved from MBP using Factor Xa as recommended (New England Biolabs) and concentrated using YM30 and YM3 microcon centrifugal filter devices (Millipore) with 30- and 3-kD cutoffs, respectively. ..

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    New England Biolabs fxa protease
    Genomic structure, protein composition, and replication of recombinant MV. (A) Plasmid p(+)MV-NSe coding for the MV antigenome (top), Pac I- Spe I fragments used for subcloning (center), and amino acid sequences (one-letter code) of the junctions between the H protein ectodomain and the specificity domains (bottom). Coding regions of the six MV cistrons are represented by solid black boxes, the transmembrane segment of the F and H proteins by a horizontally lined box, the <t>FXa</t> cleavage site and the flexible linker by a hatched box, and the ligand by a gray box. Arginine residues in parentheses have been deleted. For details see text. (B) Protein composition of recombinant viruses. Viral particles were purified by centrifugation through a 20% sucrose layer onto a 60% sucrose cushion and subsequent pelleting. The viruses were titrated, 5,000 PFU was subjected to lysis, and proteins were separated by SDS-PAGE. For immunodetection, a MV-specific antiserum was used. (C) FXa protease sensitivity of hybrid H proteins. Purified viral particles (5,000 PFU) were lysed and incubated for 1 h without (−) or with (+) FXa protease at room temperature. Proteins were separated by SDS-PAGE and detected with an H-specific antiserum. (D) FACS analysis of CD46 (interrupted line), IGF1r (dotted line), and EGFr (continuous line) expression on Vero cells. Vertical axis, cell number; horizontal axis, fluorescence intensity. The greyed profile represents incubation of the cells without the primary antibody. (E) Time course of released (left panel) and cell-associated (right panel) virus production in Vero cells infected with parental MV (diamonds), MV-H/XhEGF (squares), and <t>MV-H/XhIGF1</t> (triangles). Cells were infected at a MOI of 3 and were incubated at 32°C for the times indicated. Viral titers were determined by 50% end-point dilution. Indicated values are averages of three experiments.
    Fxa Protease, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 97/100, based on 42 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Genomic structure, protein composition, and replication of recombinant MV. (A) Plasmid p(+)MV-NSe coding for the MV antigenome (top), Pac I- Spe I fragments used for subcloning (center), and amino acid sequences (one-letter code) of the junctions between the H protein ectodomain and the specificity domains (bottom). Coding regions of the six MV cistrons are represented by solid black boxes, the transmembrane segment of the F and H proteins by a horizontally lined box, the FXa cleavage site and the flexible linker by a hatched box, and the ligand by a gray box. Arginine residues in parentheses have been deleted. For details see text. (B) Protein composition of recombinant viruses. Viral particles were purified by centrifugation through a 20% sucrose layer onto a 60% sucrose cushion and subsequent pelleting. The viruses were titrated, 5,000 PFU was subjected to lysis, and proteins were separated by SDS-PAGE. For immunodetection, a MV-specific antiserum was used. (C) FXa protease sensitivity of hybrid H proteins. Purified viral particles (5,000 PFU) were lysed and incubated for 1 h without (−) or with (+) FXa protease at room temperature. Proteins were separated by SDS-PAGE and detected with an H-specific antiserum. (D) FACS analysis of CD46 (interrupted line), IGF1r (dotted line), and EGFr (continuous line) expression on Vero cells. Vertical axis, cell number; horizontal axis, fluorescence intensity. The greyed profile represents incubation of the cells without the primary antibody. (E) Time course of released (left panel) and cell-associated (right panel) virus production in Vero cells infected with parental MV (diamonds), MV-H/XhEGF (squares), and MV-H/XhIGF1 (triangles). Cells were infected at a MOI of 3 and were incubated at 32°C for the times indicated. Viral titers were determined by 50% end-point dilution. Indicated values are averages of three experiments.

    Journal: Journal of Virology

    Article Title: Recombinant Measles Viruses Efficiently Entering Cells through Targeted Receptors

    doi:

    Figure Lengend Snippet: Genomic structure, protein composition, and replication of recombinant MV. (A) Plasmid p(+)MV-NSe coding for the MV antigenome (top), Pac I- Spe I fragments used for subcloning (center), and amino acid sequences (one-letter code) of the junctions between the H protein ectodomain and the specificity domains (bottom). Coding regions of the six MV cistrons are represented by solid black boxes, the transmembrane segment of the F and H proteins by a horizontally lined box, the FXa cleavage site and the flexible linker by a hatched box, and the ligand by a gray box. Arginine residues in parentheses have been deleted. For details see text. (B) Protein composition of recombinant viruses. Viral particles were purified by centrifugation through a 20% sucrose layer onto a 60% sucrose cushion and subsequent pelleting. The viruses were titrated, 5,000 PFU was subjected to lysis, and proteins were separated by SDS-PAGE. For immunodetection, a MV-specific antiserum was used. (C) FXa protease sensitivity of hybrid H proteins. Purified viral particles (5,000 PFU) were lysed and incubated for 1 h without (−) or with (+) FXa protease at room temperature. Proteins were separated by SDS-PAGE and detected with an H-specific antiserum. (D) FACS analysis of CD46 (interrupted line), IGF1r (dotted line), and EGFr (continuous line) expression on Vero cells. Vertical axis, cell number; horizontal axis, fluorescence intensity. The greyed profile represents incubation of the cells without the primary antibody. (E) Time course of released (left panel) and cell-associated (right panel) virus production in Vero cells infected with parental MV (diamonds), MV-H/XhEGF (squares), and MV-H/XhIGF1 (triangles). Cells were infected at a MOI of 3 and were incubated at 32°C for the times indicated. Viral titers were determined by 50% end-point dilution. Indicated values are averages of three experiments.

    Article Snippet: Analogously, proteolytic cleavage of MV-H/XhIGF1 virus particles with FXa protease resulted in the loss of more than 80% of the infectivity selectively in 3T3-hIGF1r cells (Fig. B, right panel, compare black and white columns).

    Techniques: Recombinant, Plasmid Preparation, Subcloning, Purification, Centrifugation, Lysis, SDS Page, Immunodetection, Incubation, FACS, Expressing, Fluorescence, Infection

    Competition of viral entry by soluble receptors (A) and the effect of proteolytic cleavage of the specificity domain on entry (B). (A) Competition of viral infections by soluble EGF (sEGF; top panel) or soluble IGF1 (sIGF1; bottom panel). CHO-hEGFr and Vero cells pretreated or not with sEGF were infected with MV green or MV green -H/XhEGF at a MOI of 0.3. 3T3-hIGF1r and Vero cells pretreated or not with sIGF1 were infected with MV green and MV green -H/XhIGF1 at a MOI of 0.1. The number of infected cells was determined at 24 h p.i. by counting GFP-expressing cells in a standard area. Percentages are relative to the number of GFP-expressing cells in infections without added soluble receptor. Results obtained with MV Edmonston on Vero cells are indicated by gray columns. Results obtained with recombinant MV on Vero cells are indicated by white columns and on rodent cells by black columns. (B) Effects of the treatment of recombinant viruses with FXa protease prior to infection. Vero and CHO-hEGFr or Vero and 3T3-hIGF1r cells were infected with MV green -H/XhEGF or MV green -H/XhIGF1 (10 4 PFU/well), respectively, and were pretreated with 0, 5, or 50 μg of FXa protease/ml. The number of GFP-expressing cells was determined 42 h p.i. Percentages refer to the number of cells infected with untreated virus. For details, see text.

    Journal: Journal of Virology

    Article Title: Recombinant Measles Viruses Efficiently Entering Cells through Targeted Receptors

    doi:

    Figure Lengend Snippet: Competition of viral entry by soluble receptors (A) and the effect of proteolytic cleavage of the specificity domain on entry (B). (A) Competition of viral infections by soluble EGF (sEGF; top panel) or soluble IGF1 (sIGF1; bottom panel). CHO-hEGFr and Vero cells pretreated or not with sEGF were infected with MV green or MV green -H/XhEGF at a MOI of 0.3. 3T3-hIGF1r and Vero cells pretreated or not with sIGF1 were infected with MV green and MV green -H/XhIGF1 at a MOI of 0.1. The number of infected cells was determined at 24 h p.i. by counting GFP-expressing cells in a standard area. Percentages are relative to the number of GFP-expressing cells in infections without added soluble receptor. Results obtained with MV Edmonston on Vero cells are indicated by gray columns. Results obtained with recombinant MV on Vero cells are indicated by white columns and on rodent cells by black columns. (B) Effects of the treatment of recombinant viruses with FXa protease prior to infection. Vero and CHO-hEGFr or Vero and 3T3-hIGF1r cells were infected with MV green -H/XhEGF or MV green -H/XhIGF1 (10 4 PFU/well), respectively, and were pretreated with 0, 5, or 50 μg of FXa protease/ml. The number of GFP-expressing cells was determined 42 h p.i. Percentages refer to the number of cells infected with untreated virus. For details, see text.

    Article Snippet: Analogously, proteolytic cleavage of MV-H/XhIGF1 virus particles with FXa protease resulted in the loss of more than 80% of the infectivity selectively in 3T3-hIGF1r cells (Fig. B, right panel, compare black and white columns).

    Techniques: Infection, Expressing, Recombinant

    Proteolytic Removal of the CKP Prevents Infection by MV-CKPint Virus MV-CKPint was treated with FXa protease at 100 and 200 μg/mL for 2 hr. Untreated and protease-treated virus was used to infect human glioblastoma (U87), melanoma (MDAMB-435) and vero cells for 3 hr at 37°C. The number of live cells was quantified 96 hr post-infection using the trypan blue exclusion method (bar graphs). The experiments were performed in three independent repeats. Error bars represent SEM. *p

    Journal: Molecular Therapy Oncolytics

    Article Title: Using Cystine Knot Proteins as a Novel Approach to Retarget Oncolytic Measles Virus

    doi: 10.1016/j.omto.2017.09.005

    Figure Lengend Snippet: Proteolytic Removal of the CKP Prevents Infection by MV-CKPint Virus MV-CKPint was treated with FXa protease at 100 and 200 μg/mL for 2 hr. Untreated and protease-treated virus was used to infect human glioblastoma (U87), melanoma (MDAMB-435) and vero cells for 3 hr at 37°C. The number of live cells was quantified 96 hr post-infection using the trypan blue exclusion method (bar graphs). The experiments were performed in three independent repeats. Error bars represent SEM. *p

    Article Snippet: For the FXa proteolysis assay, prior to cellular infection, the number of MV-CKPint virus equivalent to an MOI of 1 was incubated with FXa protease (New England Biolabs, Ipswich, MA, USA) at final concentrations of 100 and 200 μg/mL in Opti-MEM media.

    Techniques: Infection

    Schematic diagram of the topology of Gwt1p. A , summary of the results of glycosylation ( Glyco ) mapping and fXa cleavage analysis. Four conserved regions ( A–D ) and essential residues (Asp-145, Lys-155, Asp-165, and Arg-216) are also shown. Asp-145

    Journal: The Journal of Biological Chemistry

    Article Title:

    doi: 10.1074/jbc.M110.193490

    Figure Lengend Snippet: Schematic diagram of the topology of Gwt1p. A , summary of the results of glycosylation ( Glyco ) mapping and fXa cleavage analysis. Four conserved regions ( A–D ) and essential residues (Asp-145, Lys-155, Asp-165, and Arg-216) are also shown. Asp-145

    Article Snippet: When fXa cleavage sites were inserted into loop 5–6 ( 6 ), loop 7–8 ( 9 ), or loop 9–10 ( 12 ) of Gwt1p (see also A ), the fXa protease was accessible to the fusion proteins in the presence or absence of digitonin, suggesting that these positions were located on the cytosolic side of the ER membrane.

    Techniques:

    Determination of membrane topology of Gwt1p by factor Xa protease cleavage analysis. Membrane fractions from GC1 cells expressing wild-type ( WT ) or Gwt1p fusion proteins with fXa cleavage sequences, tagged with an HA epitope, were prepared and digested

    Journal: The Journal of Biological Chemistry

    Article Title:

    doi: 10.1074/jbc.M110.193490

    Figure Lengend Snippet: Determination of membrane topology of Gwt1p by factor Xa protease cleavage analysis. Membrane fractions from GC1 cells expressing wild-type ( WT ) or Gwt1p fusion proteins with fXa cleavage sequences, tagged with an HA epitope, were prepared and digested

    Article Snippet: When fXa cleavage sites were inserted into loop 5–6 ( 6 ), loop 7–8 ( 9 ), or loop 9–10 ( 12 ) of Gwt1p (see also A ), the fXa protease was accessible to the fusion proteins in the presence or absence of digitonin, suggesting that these positions were located on the cytosolic side of the ER membrane.

    Techniques: Expressing