cd55 expression  (Sino Biological)


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    Name:
    CD55 DAF cDNA ORF Clone Human N Myc tag
    Description:
    Full length Clone DNA of Human CD55 molecule decay accelerating factor for complement Cromer blood group transcript variant 1 with N terminal Myc tag
    Catalog Number:
    hg10101-nm
    Product Aliases:
    CR cDNA ORF Clone Human, CROM cDNA ORF Clone Human, DAF cDNA ORF Clone Human, TC cDNA ORF Clone Human
    Price:
    195.0
    Applications:
    Stable or Transient mammalian expression
    Size:
    1Unit
    Category:
    cDNA Clone
    Molecule Name:
    CD55,GPI-DAF,CD55 ,DAF
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    Structured Review

    Sino Biological cd55 expression
    CD55 DAF cDNA ORF Clone Human N Myc tag
    Full length Clone DNA of Human CD55 molecule decay accelerating factor for complement Cromer blood group transcript variant 1 with N terminal Myc tag
    https://www.bioz.com/result/cd55 expression/product/Sino Biological
    Average 92 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    cd55 expression - by Bioz Stars, 2021-02
    92/100 stars

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    1) Product Images from "Human Neonatal Fc Receptor Is the Cellular Uncoating Receptor for Enterovirus B"

    Article Title: Human Neonatal Fc Receptor Is the Cellular Uncoating Receptor for Enterovirus B

    Journal: Cell

    doi: 10.1016/j.cell.2019.04.035

    Cryo-EM Structures of the Echo 6 Viral Particle and Its Complex with Attachment (CD55) or Uncoating (FcRn) Receptor (A) The density maps of free Echo 6 full particles at pH 7.4 (2.9 Å) and pH 5.5 (3.4 Å). (B) The close-up view of the hydrophobic pocket and the “pocket factor” inside. (C and D) Cryo-EM maps (C) of Echo 6 virus in complex with CD55 at pH 7.4 (3.0 Å) and pH 5.5 (3.6 Å), as well as the close-up view (D) of the “pocket factor” with the receptor binding site shown in the same style as (B). (E–G) Cryo-EM maps (E) of Echo 6 virus in complex with FcRn at pH 7.4 (3.4 Å) and pH 5.5 (3.8 Å), as well as the close-up view showing the hydrophobic pocket of Echo 6 virus in complex with FcRn at pH 7.4 (F) and pH 5.5 (G). The receptor binding sites are shown in the same style as in (B) and (D). (H and I) Cryo-EM map of Echo 6 virus empty particle (H) at pH 7.4 (3.2 Å) and its collapsed pocket without “pocket factor” (I). All density maps are colored by radius as shown in the legend in (H). Atomic models of proteins are shown in ribbons and colored by chains with VP1, VP2, VP3, CD55, and FcRn heavy chain in green, cyan, magenta, blue, and orange, respectively. The “pocket factor” is shown in sticks, and the corresponding density map is shown at the 18σ contour level, which is highlighted by red ovals in close-up views. In the structures of the Echo 6 virus-FcRn complex at pH 5.5 and free Echo 6 virus empty particle, the pocket factor is absent, and its supposed positions are indicated by red dashed ovals. See also Figures S5 , S6 , and Table S3 .
    Figure Legend Snippet: Cryo-EM Structures of the Echo 6 Viral Particle and Its Complex with Attachment (CD55) or Uncoating (FcRn) Receptor (A) The density maps of free Echo 6 full particles at pH 7.4 (2.9 Å) and pH 5.5 (3.4 Å). (B) The close-up view of the hydrophobic pocket and the “pocket factor” inside. (C and D) Cryo-EM maps (C) of Echo 6 virus in complex with CD55 at pH 7.4 (3.0 Å) and pH 5.5 (3.6 Å), as well as the close-up view (D) of the “pocket factor” with the receptor binding site shown in the same style as (B). (E–G) Cryo-EM maps (E) of Echo 6 virus in complex with FcRn at pH 7.4 (3.4 Å) and pH 5.5 (3.8 Å), as well as the close-up view showing the hydrophobic pocket of Echo 6 virus in complex with FcRn at pH 7.4 (F) and pH 5.5 (G). The receptor binding sites are shown in the same style as in (B) and (D). (H and I) Cryo-EM map of Echo 6 virus empty particle (H) at pH 7.4 (3.2 Å) and its collapsed pocket without “pocket factor” (I). All density maps are colored by radius as shown in the legend in (H). Atomic models of proteins are shown in ribbons and colored by chains with VP1, VP2, VP3, CD55, and FcRn heavy chain in green, cyan, magenta, blue, and orange, respectively. The “pocket factor” is shown in sticks, and the corresponding density map is shown at the 18σ contour level, which is highlighted by red ovals in close-up views. In the structures of the Echo 6 virus-FcRn complex at pH 5.5 and free Echo 6 virus empty particle, the pocket factor is absent, and its supposed positions are indicated by red dashed ovals. See also Figures S5 , S6 , and Table S3 .

    Techniques Used: Binding Assay

    Molecular Interactions between Echo 6 Virus and FcRn/CD55 Receptors (A) The atomic model of Echo 6-FcRn in the main contacting interface. The viral proteins are shown in surface models, and the receptor is represented as ribbon model. VP1, VP2, and VP3 of Echo 6 are shown in green, cyan, and magenta, respectively. The interacting FCGRT subunit of FcRn is colored in orange. The “canyon” is indicated by a red asterisk. (B) The interaction details between Echo 6 virus and FcRn receptor. The proteins are colored by chains as in (A), and the contacting residues are shown as sticks and colored by elements. Hydrogen bonds are represented by black dashed lines. (C) The atomic model of the Echo 6-CD55 in the main contacting interface. The viral proteins (colored by chains) are shown in surface models, and CD55 (blue) is represented as ribbons. The interaction is mainly mediated by the SCR3 and SCR4 domains. (D) The interaction details between Echo 6 virus and CD55 in the same style of FcRn in (B). See also Figure S7 .
    Figure Legend Snippet: Molecular Interactions between Echo 6 Virus and FcRn/CD55 Receptors (A) The atomic model of Echo 6-FcRn in the main contacting interface. The viral proteins are shown in surface models, and the receptor is represented as ribbon model. VP1, VP2, and VP3 of Echo 6 are shown in green, cyan, and magenta, respectively. The interacting FCGRT subunit of FcRn is colored in orange. The “canyon” is indicated by a red asterisk. (B) The interaction details between Echo 6 virus and FcRn receptor. The proteins are colored by chains as in (A), and the contacting residues are shown as sticks and colored by elements. Hydrogen bonds are represented by black dashed lines. (C) The atomic model of the Echo 6-CD55 in the main contacting interface. The viral proteins (colored by chains) are shown in surface models, and CD55 (blue) is represented as ribbons. The interaction is mainly mediated by the SCR3 and SCR4 domains. (D) The interaction details between Echo 6 virus and CD55 in the same style of FcRn in (B). See also Figure S7 .

    Techniques Used:

    Model of Echovirus Entry into the Cell The four structural proteins (VP1, VP2, VP3, and VP4) comprising of viral capsid are indicated in blue, dark olive green, forest green, and pigment green, respectively. The viral RNA genome is shown in orange. CD55 and FcRn are displayed in light green and sky blue, respectively. The two undefined receptor binding modes on the viral surface are labeled with question marks.
    Figure Legend Snippet: Model of Echovirus Entry into the Cell The four structural proteins (VP1, VP2, VP3, and VP4) comprising of viral capsid are indicated in blue, dark olive green, forest green, and pigment green, respectively. The viral RNA genome is shown in orange. CD55 and FcRn are displayed in light green and sky blue, respectively. The two undefined receptor binding modes on the viral surface are labeled with question marks.

    Techniques Used: Binding Assay, Labeling

    Confocal Analysis of FcRn and CD55 Expressed in HEK293T and RD Cells, Related to Figure 3 (A and C) Immunofluorescence staining of FcRn in HEK293T cells (A) and RD cells (C). FcRn is expressed both on the cell surface and in the cytoplasm. (B and D) Immunofluorescence staining of CD55 in HEK293T cells (B) and RD cells (D). CD55 was mostly expressed on the cell surface.
    Figure Legend Snippet: Confocal Analysis of FcRn and CD55 Expressed in HEK293T and RD Cells, Related to Figure 3 (A and C) Immunofluorescence staining of FcRn in HEK293T cells (A) and RD cells (C). FcRn is expressed both on the cell surface and in the cytoplasm. (B and D) Immunofluorescence staining of CD55 in HEK293T cells (B) and RD cells (D). CD55 was mostly expressed on the cell surface.

    Techniques Used: Immunofluorescence, Staining

    Comprehensive Comparison of CD55 and FcRn Binding Sites on Different Enteroviruses, Related to Figure 5 (A) Cartoon diagrams of CD55 bound to different enteroviruses determined by cryo-EM. Each component is represented with a unique color, and CD55 is colored in gold. (B) Superimposition of the atomic models of CD55 bound to different enteroviruses within an asymmetric unit. The icosahedral axes are shown as triangles and pentangles. The binding site of CD55 on Echo 6 virus is similar to that on CV-B3, but different from those on the Echo 7 and Echo 12 viruses. The “canyon” is indicated by a red asterisk. (C) Comparison of the binding sites of CD55 and FcRn on Echo 6 virus. The viral proteins are shown in surface models, and the receptors are shown as ribbons. The position of the “canyon” is indicated by a red asterisk. The steric clash between CD55 and FcRn is highlighted by a dashed oval. (D) Comparison of the binding sites of CD55 and FcRn on Echo 7 virus. The two receptors could bind simultaneously on the virus without clashing. Two different views of the superimposition are shown to reveal the compatibility of the two receptors in space. VP1, VP2 and VP3 of the virus are shown in green, cyan and magenta, respectively. CD55 molecules bound to Echo 6, Echo 7, Echo 12 and CV-B3 are shown in blue, black, pink and red, respectively. The FCGRT subunit of FcRn is shown in orange, and β2 m is in gray.
    Figure Legend Snippet: Comprehensive Comparison of CD55 and FcRn Binding Sites on Different Enteroviruses, Related to Figure 5 (A) Cartoon diagrams of CD55 bound to different enteroviruses determined by cryo-EM. Each component is represented with a unique color, and CD55 is colored in gold. (B) Superimposition of the atomic models of CD55 bound to different enteroviruses within an asymmetric unit. The icosahedral axes are shown as triangles and pentangles. The binding site of CD55 on Echo 6 virus is similar to that on CV-B3, but different from those on the Echo 7 and Echo 12 viruses. The “canyon” is indicated by a red asterisk. (C) Comparison of the binding sites of CD55 and FcRn on Echo 6 virus. The viral proteins are shown in surface models, and the receptors are shown as ribbons. The position of the “canyon” is indicated by a red asterisk. The steric clash between CD55 and FcRn is highlighted by a dashed oval. (D) Comparison of the binding sites of CD55 and FcRn on Echo 7 virus. The two receptors could bind simultaneously on the virus without clashing. Two different views of the superimposition are shown to reveal the compatibility of the two receptors in space. VP1, VP2 and VP3 of the virus are shown in green, cyan and magenta, respectively. CD55 molecules bound to Echo 6, Echo 7, Echo 12 and CV-B3 are shown in blue, black, pink and red, respectively. The FCGRT subunit of FcRn is shown in orange, and β2 m is in gray.

    Techniques Used: Binding Assay

    Cryo-EM Analysis of Echo 6 Virus and Its Complex with Receptors, Related to Figure 4 (A) Gold-standard Fourier shell correlation (FSC) curves of the structures of Echo 6 virus alone or in complex with its receptors. The 0.143 cut-off value is shown to indicate the resolution of each reconstruction. (B) Local resolution maps of representative density maps of Echo 6 virus or its complex with receptors. In all of these structures, most regions reach 3.0 Å and allowed the atomic details to be resolved. (C-K) Representative density maps and atomic models of the pocket region or receptor binding interface. Most side chains of key residues within the pocket are clearly resolved. The “pocket factor” is preserved in structures of free Echo 6 full-particle or in complex with CD55 at both pH 7.4 and pH 5.5. In the structures with FcRn binding, it is well accommodated at pH 7.4, but is released at pH 5.5.
    Figure Legend Snippet: Cryo-EM Analysis of Echo 6 Virus and Its Complex with Receptors, Related to Figure 4 (A) Gold-standard Fourier shell correlation (FSC) curves of the structures of Echo 6 virus alone or in complex with its receptors. The 0.143 cut-off value is shown to indicate the resolution of each reconstruction. (B) Local resolution maps of representative density maps of Echo 6 virus or its complex with receptors. In all of these structures, most regions reach 3.0 Å and allowed the atomic details to be resolved. (C-K) Representative density maps and atomic models of the pocket region or receptor binding interface. Most side chains of key residues within the pocket are clearly resolved. The “pocket factor” is preserved in structures of free Echo 6 full-particle or in complex with CD55 at both pH 7.4 and pH 5.5. In the structures with FcRn binding, it is well accommodated at pH 7.4, but is released at pH 5.5.

    Techniques Used: Binding Assay

    Direct Binding of FcRn to Echo 6 Virus (A and B) BIAcore diagram of soluble FcRn protein (A) or CD55 protein (B) bound to Echo 6 virus. The K D values were calculated by the BIAcore 3000 analysis software (BIAevaluation version 4.1). (C and D) The binding affinity of CV-B4 virus with FcRn (C) or CD55 protein (D) was measured by SPR, which served as a control. (E–G) Binding of FcRn (E), CD55 (F), or CD26 (G, control) in 2-fold serial dilutions with Echo 6 virus was assayed using ELISAs. (H) Dose dependence of soluble FcRn or CD55 protein to block Echo 6 virus infection. CD26 was used as a control protein. (I and J) Echo 6 was incubated with FCGRT KO , B2M KO , CD55 KO , or WT HEK293T cells at 4°C for 1 h (I) or plus 37°C for 30 min (J). Cells were collected, and RNA was extracted for qPCR analysis. The data depict means with SEM. ∗∗ p
    Figure Legend Snippet: Direct Binding of FcRn to Echo 6 Virus (A and B) BIAcore diagram of soluble FcRn protein (A) or CD55 protein (B) bound to Echo 6 virus. The K D values were calculated by the BIAcore 3000 analysis software (BIAevaluation version 4.1). (C and D) The binding affinity of CV-B4 virus with FcRn (C) or CD55 protein (D) was measured by SPR, which served as a control. (E–G) Binding of FcRn (E), CD55 (F), or CD26 (G, control) in 2-fold serial dilutions with Echo 6 virus was assayed using ELISAs. (H) Dose dependence of soluble FcRn or CD55 protein to block Echo 6 virus infection. CD26 was used as a control protein. (I and J) Echo 6 was incubated with FCGRT KO , B2M KO , CD55 KO , or WT HEK293T cells at 4°C for 1 h (I) or plus 37°C for 30 min (J). Cells were collected, and RNA was extracted for qPCR analysis. The data depict means with SEM. ∗∗ p

    Techniques Used: Binding Assay, Software, SPR Assay, Blocking Assay, Infection, Incubation, Real-time Polymerase Chain Reaction

    FcRn Complementation Can Restore Susceptibility; Ectopic Expression Makes Non-permissive Cells Susceptible to Echovirus, Related to Figure 1 (A and B) western blot of FCGRT (A) and β2 m (B) expression in WT, KO and supplementary cells. (C and D) qPCR analysis of Echo 6 infection in FCGRT KO cells complemented with lentivirus expressing FCGRT (C). B2M KO cells complemented with lentivirus expressing β2 m (D). KO cell lines with lentivirus expressing GFP were used as a control. Cells were infected with 0.01 MOI Echo 6, and supernatants were taken for virus titration after 24 h. (E and F) western blot of FCGRT and CD55 expression in WT, human FcRn, human CD55 and human FcRn+ CD55 ectopic expression CHO (E) or BHK (F) cells. (G and H) qPCR analysis of Echo 6 in non-susceptible CHO (G) and BHK (H) cells with ectopic expression of human FcRn (FCGRT+B2M), CD55 or both. (I) Echovirus infection in CHO cells with the ectopic expression of FCGRT only. (The data depict means with SEM. C, D, G and H: one-way ANOVA with multiple comparisons; I: unpaired two-tailed t test, with a Welch post-correction. # stands for undetected. ∗∗∗∗ p
    Figure Legend Snippet: FcRn Complementation Can Restore Susceptibility; Ectopic Expression Makes Non-permissive Cells Susceptible to Echovirus, Related to Figure 1 (A and B) western blot of FCGRT (A) and β2 m (B) expression in WT, KO and supplementary cells. (C and D) qPCR analysis of Echo 6 infection in FCGRT KO cells complemented with lentivirus expressing FCGRT (C). B2M KO cells complemented with lentivirus expressing β2 m (D). KO cell lines with lentivirus expressing GFP were used as a control. Cells were infected with 0.01 MOI Echo 6, and supernatants were taken for virus titration after 24 h. (E and F) western blot of FCGRT and CD55 expression in WT, human FcRn, human CD55 and human FcRn+ CD55 ectopic expression CHO (E) or BHK (F) cells. (G and H) qPCR analysis of Echo 6 in non-susceptible CHO (G) and BHK (H) cells with ectopic expression of human FcRn (FCGRT+B2M), CD55 or both. (I) Echovirus infection in CHO cells with the ectopic expression of FCGRT only. (The data depict means with SEM. C, D, G and H: one-way ANOVA with multiple comparisons; I: unpaired two-tailed t test, with a Welch post-correction. # stands for undetected. ∗∗∗∗ p

    Techniques Used: Expressing, Western Blot, Real-time Polymerase Chain Reaction, Infection, Titration, Two Tailed Test

    FcRn-Decorated Liposomes Induce Echo 6 Uncoating Negative staining EM detection of: (A) Purified Echo 6 full particles (upper left) and Echo 6-CD55 complexes. (B) Echo 6-FcRn complexes. (C) Nickel-charged liposomes. (D) Echo 6 incubated with CD55-decorated liposomes. Red arrows indicate the particles that attached to the surface of the liposomes. (E) Echo 6 incubated with FcRn-decorated liposomes. Green arrows indicate empty particles. (F) Echo 6 incubated with nickel-charged liposomes. All samples were treated for 10 min at 37°C, pH 5.5. The scale bar represents 100 nm.
    Figure Legend Snippet: FcRn-Decorated Liposomes Induce Echo 6 Uncoating Negative staining EM detection of: (A) Purified Echo 6 full particles (upper left) and Echo 6-CD55 complexes. (B) Echo 6-FcRn complexes. (C) Nickel-charged liposomes. (D) Echo 6 incubated with CD55-decorated liposomes. Red arrows indicate the particles that attached to the surface of the liposomes. (E) Echo 6 incubated with FcRn-decorated liposomes. Green arrows indicate empty particles. (F) Echo 6 incubated with nickel-charged liposomes. All samples were treated for 10 min at 37°C, pH 5.5. The scale bar represents 100 nm.

    Techniques Used: Negative Staining, Purification, Incubation

    FCGRT or B2M KO Has No Effect on CD55 Expression, Related to Figure 1 (A) Diagram of FcRn. The α chain expressed by FCGRT contains a transmembrane domain and three extracellular domains (α1, α2 and α3). The β2 m chain is expressed by B2M . (B) Sanger sequencing of FCGRT, B2M and CD55 in KO cells. Sequencing data shows an alignment of WT sequence with the KO sequence. (C) western blot of CD55 expression in WT, FCGRT KO , B2M KO and CD55 KO cells. (D) Flow cytometry analysis of CD55 expression in WT, FCGRT KO , B2M KO (left panel: fixed cell staining, middle panel: cell surface expression) and CD55 KO cells (right panel).
    Figure Legend Snippet: FCGRT or B2M KO Has No Effect on CD55 Expression, Related to Figure 1 (A) Diagram of FcRn. The α chain expressed by FCGRT contains a transmembrane domain and three extracellular domains (α1, α2 and α3). The β2 m chain is expressed by B2M . (B) Sanger sequencing of FCGRT, B2M and CD55 in KO cells. Sequencing data shows an alignment of WT sequence with the KO sequence. (C) western blot of CD55 expression in WT, FCGRT KO , B2M KO and CD55 KO cells. (D) Flow cytometry analysis of CD55 expression in WT, FCGRT KO , B2M KO (left panel: fixed cell staining, middle panel: cell surface expression) and CD55 KO cells (right panel).

    Techniques Used: Expressing, Sequencing, Western Blot, Flow Cytometry, Staining

    CRISPR Screening Uncovered FcRn, a Functional Receptor for Echo 6 Infection (A) Dot plot illustrating the enriched genes from the CRISPR screen. Genes were rank-ordered by robust rank aggregation (RRA) scores based on MAGeCK analysis. The height and size of the top three genes, differently colored, was proportional to their RRA scores. (B) Schematic diagram of sgRNA library construction and Echo 6 receptor screening. (C and G) Echo 6 infection in FCGRT KO , B2M KO , CD55 KO , and wild type (WT) HEK293T cells (C). Echo 6 infection in CD55 KO (G), with the above three cell lines in (C) as controls. TCID 50 was calculated at 48 h post infection (h.p.i). “+” and “-” indicate the presence or absence of FcRn or CD55 in the KO cell lines. (D and E) Echo 6 infection in FCGRT KO cells complemented with lentivirus expressing FCGRT (D) and B2M KO cells complemented with lentivirus expressing β2 m (E). KO cell lines with lentivirus expressing GFP was used as a control. Cells were infected with Echo 6 (MOI 0.01), and supernatants were harvested for virus titration at 24 h.p.i. (F) Light microscopy images showing CPE of FCGRT KO , B2M KO , CD55 KO , and WT HEK293T cells at 24 h.p.i with Echo 6 (MOI 1). Immunofluorescence images of KO and WT cells infected by Echo 6 (MOI 10) for 8 h and then stained by anti-Echo 6 antibodies. The scale bar represents 50 μm. (H) Growth curve of Echo 6 in FCGRT KO , B2M KO , CD55 KO , and WT HEK293T cells. (I) Echo 6 was inoculated onto non-susceptible CHO (left) and BHK (right) cells with ectopic expression of human FcRn (FCGRT+β2 m), CD55, or both. The data depict means with SEM. TCID 50 values were calculated by the Reed-Muench method. Experiments were repeated three times. # stands for undetected; ∗∗∗∗ p
    Figure Legend Snippet: CRISPR Screening Uncovered FcRn, a Functional Receptor for Echo 6 Infection (A) Dot plot illustrating the enriched genes from the CRISPR screen. Genes were rank-ordered by robust rank aggregation (RRA) scores based on MAGeCK analysis. The height and size of the top three genes, differently colored, was proportional to their RRA scores. (B) Schematic diagram of sgRNA library construction and Echo 6 receptor screening. (C and G) Echo 6 infection in FCGRT KO , B2M KO , CD55 KO , and wild type (WT) HEK293T cells (C). Echo 6 infection in CD55 KO (G), with the above three cell lines in (C) as controls. TCID 50 was calculated at 48 h post infection (h.p.i). “+” and “-” indicate the presence or absence of FcRn or CD55 in the KO cell lines. (D and E) Echo 6 infection in FCGRT KO cells complemented with lentivirus expressing FCGRT (D) and B2M KO cells complemented with lentivirus expressing β2 m (E). KO cell lines with lentivirus expressing GFP was used as a control. Cells were infected with Echo 6 (MOI 0.01), and supernatants were harvested for virus titration at 24 h.p.i. (F) Light microscopy images showing CPE of FCGRT KO , B2M KO , CD55 KO , and WT HEK293T cells at 24 h.p.i with Echo 6 (MOI 1). Immunofluorescence images of KO and WT cells infected by Echo 6 (MOI 10) for 8 h and then stained by anti-Echo 6 antibodies. The scale bar represents 50 μm. (H) Growth curve of Echo 6 in FCGRT KO , B2M KO , CD55 KO , and WT HEK293T cells. (I) Echo 6 was inoculated onto non-susceptible CHO (left) and BHK (right) cells with ectopic expression of human FcRn (FCGRT+β2 m), CD55, or both. The data depict means with SEM. TCID 50 values were calculated by the Reed-Muench method. Experiments were repeated three times. # stands for undetected; ∗∗∗∗ p

    Techniques Used: CRISPR, Functional Assay, Infection, Expressing, Titration, Light Microscopy, Immunofluorescence, Staining, Endpoint Dilution Assay

    FcRn and CD55 Soluble Protein Expression and Purification, Related to Figure 3 (A) FcRn soluble protein was expressed by co-transfection of constructs encoding the FCGRT extracellular domain and B2M in HEK293T cells. The protein was eluted as a single peak from gel filtration on Superdex 200 column (GE Healthcare), and two bands showing FCGRT and B2M were evident by SDS-PAGE. (B) Gel filtration and SDS-PAGE of CD55 soluble protein purification.
    Figure Legend Snippet: FcRn and CD55 Soluble Protein Expression and Purification, Related to Figure 3 (A) FcRn soluble protein was expressed by co-transfection of constructs encoding the FCGRT extracellular domain and B2M in HEK293T cells. The protein was eluted as a single peak from gel filtration on Superdex 200 column (GE Healthcare), and two bands showing FCGRT and B2M were evident by SDS-PAGE. (B) Gel filtration and SDS-PAGE of CD55 soluble protein purification.

    Techniques Used: Expressing, Purification, Cotransfection, Construct, Filtration, SDS Page, Protein Purification

    Related Articles

    Transfection:

    Article Title: Human Neonatal Fc Receptor Is the Cellular Uncoating Receptor for Enterovirus B
    Article Snippet: .. Plasmid expressing human CD55 was transfected into HEK293T cells for CD55 expression. .. In each case, the supernatant was collected, and the protein was subsequently purified by Ni-nitrilotriacetic acid (NTA) chromatography (GE Healthcare) and gel filtration using a Hiload 16/600 Superdex 200 PG column (GE Healthcare) with a buffer containing 20 mM Tris·HCl and 150 mM NaCl (pH 8.0).

    Article Title: Human Neonatal Fc Receptor Is the Cellular Uncoating Receptor for Enterovirus B.
    Article Snippet: .. Plasmid expressing human CD55 was transfected into HEK293T cells for CD55 expression. .. In each case, the supernatant was collected, and the protein was subsequently purified by Ni-nitrilotriacetic acid (NTA) chromatography (GE Healthcare) and gel filtration using a Hiload 16/600 Superdex 200 PG column (GE Healthcare) with a buffer containing 20 mM Tris$HCl and 150 mM NaCl (pH 8.0).

    Plasmid Preparation:

    Article Title: Human Neonatal Fc Receptor Is the Cellular Uncoating Receptor for Enterovirus B
    Article Snippet: .. Plasmid expressing human CD55 was transfected into HEK293T cells for CD55 expression. .. In each case, the supernatant was collected, and the protein was subsequently purified by Ni-nitrilotriacetic acid (NTA) chromatography (GE Healthcare) and gel filtration using a Hiload 16/600 Superdex 200 PG column (GE Healthcare) with a buffer containing 20 mM Tris·HCl and 150 mM NaCl (pH 8.0).

    Article Title: Human Neonatal Fc Receptor Is the Cellular Uncoating Receptor for Enterovirus B.
    Article Snippet: .. Plasmid expressing human CD55 was transfected into HEK293T cells for CD55 expression. .. In each case, the supernatant was collected, and the protein was subsequently purified by Ni-nitrilotriacetic acid (NTA) chromatography (GE Healthcare) and gel filtration using a Hiload 16/600 Superdex 200 PG column (GE Healthcare) with a buffer containing 20 mM Tris$HCl and 150 mM NaCl (pH 8.0).

    Expressing:

    Article Title: Human Neonatal Fc Receptor Is the Cellular Uncoating Receptor for Enterovirus B
    Article Snippet: .. Plasmid expressing human CD55 was transfected into HEK293T cells for CD55 expression. .. In each case, the supernatant was collected, and the protein was subsequently purified by Ni-nitrilotriacetic acid (NTA) chromatography (GE Healthcare) and gel filtration using a Hiload 16/600 Superdex 200 PG column (GE Healthcare) with a buffer containing 20 mM Tris·HCl and 150 mM NaCl (pH 8.0).

    Article Title: Human Neonatal Fc Receptor Is the Cellular Uncoating Receptor for Enterovirus B.
    Article Snippet: .. Plasmid expressing human CD55 was transfected into HEK293T cells for CD55 expression. .. In each case, the supernatant was collected, and the protein was subsequently purified by Ni-nitrilotriacetic acid (NTA) chromatography (GE Healthcare) and gel filtration using a Hiload 16/600 Superdex 200 PG column (GE Healthcare) with a buffer containing 20 mM Tris$HCl and 150 mM NaCl (pH 8.0).

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    Sino Biological cd55 expression
    Cryo-EM Structures of the Echo 6 Viral Particle and Its Complex with Attachment <t>(CD55)</t> or Uncoating (FcRn) Receptor (A) The density maps of free Echo 6 full particles at pH 7.4 (2.9 Å) and pH 5.5 (3.4 Å). (B) The close-up view of the hydrophobic pocket and the “pocket factor” inside. (C and D) Cryo-EM maps (C) of Echo 6 virus in complex with CD55 at pH 7.4 (3.0 Å) and pH 5.5 (3.6 Å), as well as the close-up view (D) of the “pocket factor” with the receptor binding site shown in the same style as (B). (E–G) Cryo-EM maps (E) of Echo 6 virus in complex with FcRn at pH 7.4 (3.4 Å) and pH 5.5 (3.8 Å), as well as the close-up view showing the hydrophobic pocket of Echo 6 virus in complex with FcRn at pH 7.4 (F) and pH 5.5 (G). The receptor binding sites are shown in the same style as in (B) and (D). (H and I) Cryo-EM map of Echo 6 virus empty particle (H) at pH 7.4 (3.2 Å) and its collapsed pocket without “pocket factor” (I). All density maps are colored by radius as shown in the legend in (H). Atomic models of proteins are shown in ribbons and colored by chains with VP1, VP2, VP3, CD55, and FcRn heavy chain in green, cyan, magenta, blue, and orange, respectively. The “pocket factor” is shown in sticks, and the corresponding density map is shown at the 18σ contour level, which is highlighted by red ovals in close-up views. In the structures of the Echo 6 virus-FcRn complex at pH 5.5 and free Echo 6 virus empty particle, the pocket factor is absent, and its supposed positions are indicated by red dashed ovals. See also Figures S5 , S6 , and Table S3 .
    Cd55 Expression, supplied by Sino Biological, used in various techniques. Bioz Stars score: 92/100, based on 0 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Cryo-EM Structures of the Echo 6 Viral Particle and Its Complex with Attachment (CD55) or Uncoating (FcRn) Receptor (A) The density maps of free Echo 6 full particles at pH 7.4 (2.9 Å) and pH 5.5 (3.4 Å). (B) The close-up view of the hydrophobic pocket and the “pocket factor” inside. (C and D) Cryo-EM maps (C) of Echo 6 virus in complex with CD55 at pH 7.4 (3.0 Å) and pH 5.5 (3.6 Å), as well as the close-up view (D) of the “pocket factor” with the receptor binding site shown in the same style as (B). (E–G) Cryo-EM maps (E) of Echo 6 virus in complex with FcRn at pH 7.4 (3.4 Å) and pH 5.5 (3.8 Å), as well as the close-up view showing the hydrophobic pocket of Echo 6 virus in complex with FcRn at pH 7.4 (F) and pH 5.5 (G). The receptor binding sites are shown in the same style as in (B) and (D). (H and I) Cryo-EM map of Echo 6 virus empty particle (H) at pH 7.4 (3.2 Å) and its collapsed pocket without “pocket factor” (I). All density maps are colored by radius as shown in the legend in (H). Atomic models of proteins are shown in ribbons and colored by chains with VP1, VP2, VP3, CD55, and FcRn heavy chain in green, cyan, magenta, blue, and orange, respectively. The “pocket factor” is shown in sticks, and the corresponding density map is shown at the 18σ contour level, which is highlighted by red ovals in close-up views. In the structures of the Echo 6 virus-FcRn complex at pH 5.5 and free Echo 6 virus empty particle, the pocket factor is absent, and its supposed positions are indicated by red dashed ovals. See also Figures S5 , S6 , and Table S3 .

    Journal: Cell

    Article Title: Human Neonatal Fc Receptor Is the Cellular Uncoating Receptor for Enterovirus B

    doi: 10.1016/j.cell.2019.04.035

    Figure Lengend Snippet: Cryo-EM Structures of the Echo 6 Viral Particle and Its Complex with Attachment (CD55) or Uncoating (FcRn) Receptor (A) The density maps of free Echo 6 full particles at pH 7.4 (2.9 Å) and pH 5.5 (3.4 Å). (B) The close-up view of the hydrophobic pocket and the “pocket factor” inside. (C and D) Cryo-EM maps (C) of Echo 6 virus in complex with CD55 at pH 7.4 (3.0 Å) and pH 5.5 (3.6 Å), as well as the close-up view (D) of the “pocket factor” with the receptor binding site shown in the same style as (B). (E–G) Cryo-EM maps (E) of Echo 6 virus in complex with FcRn at pH 7.4 (3.4 Å) and pH 5.5 (3.8 Å), as well as the close-up view showing the hydrophobic pocket of Echo 6 virus in complex with FcRn at pH 7.4 (F) and pH 5.5 (G). The receptor binding sites are shown in the same style as in (B) and (D). (H and I) Cryo-EM map of Echo 6 virus empty particle (H) at pH 7.4 (3.2 Å) and its collapsed pocket without “pocket factor” (I). All density maps are colored by radius as shown in the legend in (H). Atomic models of proteins are shown in ribbons and colored by chains with VP1, VP2, VP3, CD55, and FcRn heavy chain in green, cyan, magenta, blue, and orange, respectively. The “pocket factor” is shown in sticks, and the corresponding density map is shown at the 18σ contour level, which is highlighted by red ovals in close-up views. In the structures of the Echo 6 virus-FcRn complex at pH 5.5 and free Echo 6 virus empty particle, the pocket factor is absent, and its supposed positions are indicated by red dashed ovals. See also Figures S5 , S6 , and Table S3 .

    Article Snippet: Plasmid expressing human CD55 was transfected into HEK293T cells for CD55 expression.

    Techniques: Binding Assay

    Molecular Interactions between Echo 6 Virus and FcRn/CD55 Receptors (A) The atomic model of Echo 6-FcRn in the main contacting interface. The viral proteins are shown in surface models, and the receptor is represented as ribbon model. VP1, VP2, and VP3 of Echo 6 are shown in green, cyan, and magenta, respectively. The interacting FCGRT subunit of FcRn is colored in orange. The “canyon” is indicated by a red asterisk. (B) The interaction details between Echo 6 virus and FcRn receptor. The proteins are colored by chains as in (A), and the contacting residues are shown as sticks and colored by elements. Hydrogen bonds are represented by black dashed lines. (C) The atomic model of the Echo 6-CD55 in the main contacting interface. The viral proteins (colored by chains) are shown in surface models, and CD55 (blue) is represented as ribbons. The interaction is mainly mediated by the SCR3 and SCR4 domains. (D) The interaction details between Echo 6 virus and CD55 in the same style of FcRn in (B). See also Figure S7 .

    Journal: Cell

    Article Title: Human Neonatal Fc Receptor Is the Cellular Uncoating Receptor for Enterovirus B

    doi: 10.1016/j.cell.2019.04.035

    Figure Lengend Snippet: Molecular Interactions between Echo 6 Virus and FcRn/CD55 Receptors (A) The atomic model of Echo 6-FcRn in the main contacting interface. The viral proteins are shown in surface models, and the receptor is represented as ribbon model. VP1, VP2, and VP3 of Echo 6 are shown in green, cyan, and magenta, respectively. The interacting FCGRT subunit of FcRn is colored in orange. The “canyon” is indicated by a red asterisk. (B) The interaction details between Echo 6 virus and FcRn receptor. The proteins are colored by chains as in (A), and the contacting residues are shown as sticks and colored by elements. Hydrogen bonds are represented by black dashed lines. (C) The atomic model of the Echo 6-CD55 in the main contacting interface. The viral proteins (colored by chains) are shown in surface models, and CD55 (blue) is represented as ribbons. The interaction is mainly mediated by the SCR3 and SCR4 domains. (D) The interaction details between Echo 6 virus and CD55 in the same style of FcRn in (B). See also Figure S7 .

    Article Snippet: Plasmid expressing human CD55 was transfected into HEK293T cells for CD55 expression.

    Techniques:

    Model of Echovirus Entry into the Cell The four structural proteins (VP1, VP2, VP3, and VP4) comprising of viral capsid are indicated in blue, dark olive green, forest green, and pigment green, respectively. The viral RNA genome is shown in orange. CD55 and FcRn are displayed in light green and sky blue, respectively. The two undefined receptor binding modes on the viral surface are labeled with question marks.

    Journal: Cell

    Article Title: Human Neonatal Fc Receptor Is the Cellular Uncoating Receptor for Enterovirus B

    doi: 10.1016/j.cell.2019.04.035

    Figure Lengend Snippet: Model of Echovirus Entry into the Cell The four structural proteins (VP1, VP2, VP3, and VP4) comprising of viral capsid are indicated in blue, dark olive green, forest green, and pigment green, respectively. The viral RNA genome is shown in orange. CD55 and FcRn are displayed in light green and sky blue, respectively. The two undefined receptor binding modes on the viral surface are labeled with question marks.

    Article Snippet: Plasmid expressing human CD55 was transfected into HEK293T cells for CD55 expression.

    Techniques: Binding Assay, Labeling

    Confocal Analysis of FcRn and CD55 Expressed in HEK293T and RD Cells, Related to Figure 3 (A and C) Immunofluorescence staining of FcRn in HEK293T cells (A) and RD cells (C). FcRn is expressed both on the cell surface and in the cytoplasm. (B and D) Immunofluorescence staining of CD55 in HEK293T cells (B) and RD cells (D). CD55 was mostly expressed on the cell surface.

    Journal: Cell

    Article Title: Human Neonatal Fc Receptor Is the Cellular Uncoating Receptor for Enterovirus B

    doi: 10.1016/j.cell.2019.04.035

    Figure Lengend Snippet: Confocal Analysis of FcRn and CD55 Expressed in HEK293T and RD Cells, Related to Figure 3 (A and C) Immunofluorescence staining of FcRn in HEK293T cells (A) and RD cells (C). FcRn is expressed both on the cell surface and in the cytoplasm. (B and D) Immunofluorescence staining of CD55 in HEK293T cells (B) and RD cells (D). CD55 was mostly expressed on the cell surface.

    Article Snippet: Plasmid expressing human CD55 was transfected into HEK293T cells for CD55 expression.

    Techniques: Immunofluorescence, Staining

    Comprehensive Comparison of CD55 and FcRn Binding Sites on Different Enteroviruses, Related to Figure 5 (A) Cartoon diagrams of CD55 bound to different enteroviruses determined by cryo-EM. Each component is represented with a unique color, and CD55 is colored in gold. (B) Superimposition of the atomic models of CD55 bound to different enteroviruses within an asymmetric unit. The icosahedral axes are shown as triangles and pentangles. The binding site of CD55 on Echo 6 virus is similar to that on CV-B3, but different from those on the Echo 7 and Echo 12 viruses. The “canyon” is indicated by a red asterisk. (C) Comparison of the binding sites of CD55 and FcRn on Echo 6 virus. The viral proteins are shown in surface models, and the receptors are shown as ribbons. The position of the “canyon” is indicated by a red asterisk. The steric clash between CD55 and FcRn is highlighted by a dashed oval. (D) Comparison of the binding sites of CD55 and FcRn on Echo 7 virus. The two receptors could bind simultaneously on the virus without clashing. Two different views of the superimposition are shown to reveal the compatibility of the two receptors in space. VP1, VP2 and VP3 of the virus are shown in green, cyan and magenta, respectively. CD55 molecules bound to Echo 6, Echo 7, Echo 12 and CV-B3 are shown in blue, black, pink and red, respectively. The FCGRT subunit of FcRn is shown in orange, and β2 m is in gray.

    Journal: Cell

    Article Title: Human Neonatal Fc Receptor Is the Cellular Uncoating Receptor for Enterovirus B

    doi: 10.1016/j.cell.2019.04.035

    Figure Lengend Snippet: Comprehensive Comparison of CD55 and FcRn Binding Sites on Different Enteroviruses, Related to Figure 5 (A) Cartoon diagrams of CD55 bound to different enteroviruses determined by cryo-EM. Each component is represented with a unique color, and CD55 is colored in gold. (B) Superimposition of the atomic models of CD55 bound to different enteroviruses within an asymmetric unit. The icosahedral axes are shown as triangles and pentangles. The binding site of CD55 on Echo 6 virus is similar to that on CV-B3, but different from those on the Echo 7 and Echo 12 viruses. The “canyon” is indicated by a red asterisk. (C) Comparison of the binding sites of CD55 and FcRn on Echo 6 virus. The viral proteins are shown in surface models, and the receptors are shown as ribbons. The position of the “canyon” is indicated by a red asterisk. The steric clash between CD55 and FcRn is highlighted by a dashed oval. (D) Comparison of the binding sites of CD55 and FcRn on Echo 7 virus. The two receptors could bind simultaneously on the virus without clashing. Two different views of the superimposition are shown to reveal the compatibility of the two receptors in space. VP1, VP2 and VP3 of the virus are shown in green, cyan and magenta, respectively. CD55 molecules bound to Echo 6, Echo 7, Echo 12 and CV-B3 are shown in blue, black, pink and red, respectively. The FCGRT subunit of FcRn is shown in orange, and β2 m is in gray.

    Article Snippet: Plasmid expressing human CD55 was transfected into HEK293T cells for CD55 expression.

    Techniques: Binding Assay

    Cryo-EM Analysis of Echo 6 Virus and Its Complex with Receptors, Related to Figure 4 (A) Gold-standard Fourier shell correlation (FSC) curves of the structures of Echo 6 virus alone or in complex with its receptors. The 0.143 cut-off value is shown to indicate the resolution of each reconstruction. (B) Local resolution maps of representative density maps of Echo 6 virus or its complex with receptors. In all of these structures, most regions reach 3.0 Å and allowed the atomic details to be resolved. (C-K) Representative density maps and atomic models of the pocket region or receptor binding interface. Most side chains of key residues within the pocket are clearly resolved. The “pocket factor” is preserved in structures of free Echo 6 full-particle or in complex with CD55 at both pH 7.4 and pH 5.5. In the structures with FcRn binding, it is well accommodated at pH 7.4, but is released at pH 5.5.

    Journal: Cell

    Article Title: Human Neonatal Fc Receptor Is the Cellular Uncoating Receptor for Enterovirus B

    doi: 10.1016/j.cell.2019.04.035

    Figure Lengend Snippet: Cryo-EM Analysis of Echo 6 Virus and Its Complex with Receptors, Related to Figure 4 (A) Gold-standard Fourier shell correlation (FSC) curves of the structures of Echo 6 virus alone or in complex with its receptors. The 0.143 cut-off value is shown to indicate the resolution of each reconstruction. (B) Local resolution maps of representative density maps of Echo 6 virus or its complex with receptors. In all of these structures, most regions reach 3.0 Å and allowed the atomic details to be resolved. (C-K) Representative density maps and atomic models of the pocket region or receptor binding interface. Most side chains of key residues within the pocket are clearly resolved. The “pocket factor” is preserved in structures of free Echo 6 full-particle or in complex with CD55 at both pH 7.4 and pH 5.5. In the structures with FcRn binding, it is well accommodated at pH 7.4, but is released at pH 5.5.

    Article Snippet: Plasmid expressing human CD55 was transfected into HEK293T cells for CD55 expression.

    Techniques: Binding Assay

    Direct Binding of FcRn to Echo 6 Virus (A and B) BIAcore diagram of soluble FcRn protein (A) or CD55 protein (B) bound to Echo 6 virus. The K D values were calculated by the BIAcore 3000 analysis software (BIAevaluation version 4.1). (C and D) The binding affinity of CV-B4 virus with FcRn (C) or CD55 protein (D) was measured by SPR, which served as a control. (E–G) Binding of FcRn (E), CD55 (F), or CD26 (G, control) in 2-fold serial dilutions with Echo 6 virus was assayed using ELISAs. (H) Dose dependence of soluble FcRn or CD55 protein to block Echo 6 virus infection. CD26 was used as a control protein. (I and J) Echo 6 was incubated with FCGRT KO , B2M KO , CD55 KO , or WT HEK293T cells at 4°C for 1 h (I) or plus 37°C for 30 min (J). Cells were collected, and RNA was extracted for qPCR analysis. The data depict means with SEM. ∗∗ p

    Journal: Cell

    Article Title: Human Neonatal Fc Receptor Is the Cellular Uncoating Receptor for Enterovirus B

    doi: 10.1016/j.cell.2019.04.035

    Figure Lengend Snippet: Direct Binding of FcRn to Echo 6 Virus (A and B) BIAcore diagram of soluble FcRn protein (A) or CD55 protein (B) bound to Echo 6 virus. The K D values were calculated by the BIAcore 3000 analysis software (BIAevaluation version 4.1). (C and D) The binding affinity of CV-B4 virus with FcRn (C) or CD55 protein (D) was measured by SPR, which served as a control. (E–G) Binding of FcRn (E), CD55 (F), or CD26 (G, control) in 2-fold serial dilutions with Echo 6 virus was assayed using ELISAs. (H) Dose dependence of soluble FcRn or CD55 protein to block Echo 6 virus infection. CD26 was used as a control protein. (I and J) Echo 6 was incubated with FCGRT KO , B2M KO , CD55 KO , or WT HEK293T cells at 4°C for 1 h (I) or plus 37°C for 30 min (J). Cells were collected, and RNA was extracted for qPCR analysis. The data depict means with SEM. ∗∗ p

    Article Snippet: Plasmid expressing human CD55 was transfected into HEK293T cells for CD55 expression.

    Techniques: Binding Assay, Software, SPR Assay, Blocking Assay, Infection, Incubation, Real-time Polymerase Chain Reaction

    FcRn Complementation Can Restore Susceptibility; Ectopic Expression Makes Non-permissive Cells Susceptible to Echovirus, Related to Figure 1 (A and B) western blot of FCGRT (A) and β2 m (B) expression in WT, KO and supplementary cells. (C and D) qPCR analysis of Echo 6 infection in FCGRT KO cells complemented with lentivirus expressing FCGRT (C). B2M KO cells complemented with lentivirus expressing β2 m (D). KO cell lines with lentivirus expressing GFP were used as a control. Cells were infected with 0.01 MOI Echo 6, and supernatants were taken for virus titration after 24 h. (E and F) western blot of FCGRT and CD55 expression in WT, human FcRn, human CD55 and human FcRn+ CD55 ectopic expression CHO (E) or BHK (F) cells. (G and H) qPCR analysis of Echo 6 in non-susceptible CHO (G) and BHK (H) cells with ectopic expression of human FcRn (FCGRT+B2M), CD55 or both. (I) Echovirus infection in CHO cells with the ectopic expression of FCGRT only. (The data depict means with SEM. C, D, G and H: one-way ANOVA with multiple comparisons; I: unpaired two-tailed t test, with a Welch post-correction. # stands for undetected. ∗∗∗∗ p

    Journal: Cell

    Article Title: Human Neonatal Fc Receptor Is the Cellular Uncoating Receptor for Enterovirus B

    doi: 10.1016/j.cell.2019.04.035

    Figure Lengend Snippet: FcRn Complementation Can Restore Susceptibility; Ectopic Expression Makes Non-permissive Cells Susceptible to Echovirus, Related to Figure 1 (A and B) western blot of FCGRT (A) and β2 m (B) expression in WT, KO and supplementary cells. (C and D) qPCR analysis of Echo 6 infection in FCGRT KO cells complemented with lentivirus expressing FCGRT (C). B2M KO cells complemented with lentivirus expressing β2 m (D). KO cell lines with lentivirus expressing GFP were used as a control. Cells were infected with 0.01 MOI Echo 6, and supernatants were taken for virus titration after 24 h. (E and F) western blot of FCGRT and CD55 expression in WT, human FcRn, human CD55 and human FcRn+ CD55 ectopic expression CHO (E) or BHK (F) cells. (G and H) qPCR analysis of Echo 6 in non-susceptible CHO (G) and BHK (H) cells with ectopic expression of human FcRn (FCGRT+B2M), CD55 or both. (I) Echovirus infection in CHO cells with the ectopic expression of FCGRT only. (The data depict means with SEM. C, D, G and H: one-way ANOVA with multiple comparisons; I: unpaired two-tailed t test, with a Welch post-correction. # stands for undetected. ∗∗∗∗ p

    Article Snippet: Plasmid expressing human CD55 was transfected into HEK293T cells for CD55 expression.

    Techniques: Expressing, Western Blot, Real-time Polymerase Chain Reaction, Infection, Titration, Two Tailed Test

    FcRn-Decorated Liposomes Induce Echo 6 Uncoating Negative staining EM detection of: (A) Purified Echo 6 full particles (upper left) and Echo 6-CD55 complexes. (B) Echo 6-FcRn complexes. (C) Nickel-charged liposomes. (D) Echo 6 incubated with CD55-decorated liposomes. Red arrows indicate the particles that attached to the surface of the liposomes. (E) Echo 6 incubated with FcRn-decorated liposomes. Green arrows indicate empty particles. (F) Echo 6 incubated with nickel-charged liposomes. All samples were treated for 10 min at 37°C, pH 5.5. The scale bar represents 100 nm.

    Journal: Cell

    Article Title: Human Neonatal Fc Receptor Is the Cellular Uncoating Receptor for Enterovirus B

    doi: 10.1016/j.cell.2019.04.035

    Figure Lengend Snippet: FcRn-Decorated Liposomes Induce Echo 6 Uncoating Negative staining EM detection of: (A) Purified Echo 6 full particles (upper left) and Echo 6-CD55 complexes. (B) Echo 6-FcRn complexes. (C) Nickel-charged liposomes. (D) Echo 6 incubated with CD55-decorated liposomes. Red arrows indicate the particles that attached to the surface of the liposomes. (E) Echo 6 incubated with FcRn-decorated liposomes. Green arrows indicate empty particles. (F) Echo 6 incubated with nickel-charged liposomes. All samples were treated for 10 min at 37°C, pH 5.5. The scale bar represents 100 nm.

    Article Snippet: Plasmid expressing human CD55 was transfected into HEK293T cells for CD55 expression.

    Techniques: Negative Staining, Purification, Incubation

    FCGRT or B2M KO Has No Effect on CD55 Expression, Related to Figure 1 (A) Diagram of FcRn. The α chain expressed by FCGRT contains a transmembrane domain and three extracellular domains (α1, α2 and α3). The β2 m chain is expressed by B2M . (B) Sanger sequencing of FCGRT, B2M and CD55 in KO cells. Sequencing data shows an alignment of WT sequence with the KO sequence. (C) western blot of CD55 expression in WT, FCGRT KO , B2M KO and CD55 KO cells. (D) Flow cytometry analysis of CD55 expression in WT, FCGRT KO , B2M KO (left panel: fixed cell staining, middle panel: cell surface expression) and CD55 KO cells (right panel).

    Journal: Cell

    Article Title: Human Neonatal Fc Receptor Is the Cellular Uncoating Receptor for Enterovirus B

    doi: 10.1016/j.cell.2019.04.035

    Figure Lengend Snippet: FCGRT or B2M KO Has No Effect on CD55 Expression, Related to Figure 1 (A) Diagram of FcRn. The α chain expressed by FCGRT contains a transmembrane domain and three extracellular domains (α1, α2 and α3). The β2 m chain is expressed by B2M . (B) Sanger sequencing of FCGRT, B2M and CD55 in KO cells. Sequencing data shows an alignment of WT sequence with the KO sequence. (C) western blot of CD55 expression in WT, FCGRT KO , B2M KO and CD55 KO cells. (D) Flow cytometry analysis of CD55 expression in WT, FCGRT KO , B2M KO (left panel: fixed cell staining, middle panel: cell surface expression) and CD55 KO cells (right panel).

    Article Snippet: Plasmid expressing human CD55 was transfected into HEK293T cells for CD55 expression.

    Techniques: Expressing, Sequencing, Western Blot, Flow Cytometry, Staining

    CRISPR Screening Uncovered FcRn, a Functional Receptor for Echo 6 Infection (A) Dot plot illustrating the enriched genes from the CRISPR screen. Genes were rank-ordered by robust rank aggregation (RRA) scores based on MAGeCK analysis. The height and size of the top three genes, differently colored, was proportional to their RRA scores. (B) Schematic diagram of sgRNA library construction and Echo 6 receptor screening. (C and G) Echo 6 infection in FCGRT KO , B2M KO , CD55 KO , and wild type (WT) HEK293T cells (C). Echo 6 infection in CD55 KO (G), with the above three cell lines in (C) as controls. TCID 50 was calculated at 48 h post infection (h.p.i). “+” and “-” indicate the presence or absence of FcRn or CD55 in the KO cell lines. (D and E) Echo 6 infection in FCGRT KO cells complemented with lentivirus expressing FCGRT (D) and B2M KO cells complemented with lentivirus expressing β2 m (E). KO cell lines with lentivirus expressing GFP was used as a control. Cells were infected with Echo 6 (MOI 0.01), and supernatants were harvested for virus titration at 24 h.p.i. (F) Light microscopy images showing CPE of FCGRT KO , B2M KO , CD55 KO , and WT HEK293T cells at 24 h.p.i with Echo 6 (MOI 1). Immunofluorescence images of KO and WT cells infected by Echo 6 (MOI 10) for 8 h and then stained by anti-Echo 6 antibodies. The scale bar represents 50 μm. (H) Growth curve of Echo 6 in FCGRT KO , B2M KO , CD55 KO , and WT HEK293T cells. (I) Echo 6 was inoculated onto non-susceptible CHO (left) and BHK (right) cells with ectopic expression of human FcRn (FCGRT+β2 m), CD55, or both. The data depict means with SEM. TCID 50 values were calculated by the Reed-Muench method. Experiments were repeated three times. # stands for undetected; ∗∗∗∗ p

    Journal: Cell

    Article Title: Human Neonatal Fc Receptor Is the Cellular Uncoating Receptor for Enterovirus B

    doi: 10.1016/j.cell.2019.04.035

    Figure Lengend Snippet: CRISPR Screening Uncovered FcRn, a Functional Receptor for Echo 6 Infection (A) Dot plot illustrating the enriched genes from the CRISPR screen. Genes were rank-ordered by robust rank aggregation (RRA) scores based on MAGeCK analysis. The height and size of the top three genes, differently colored, was proportional to their RRA scores. (B) Schematic diagram of sgRNA library construction and Echo 6 receptor screening. (C and G) Echo 6 infection in FCGRT KO , B2M KO , CD55 KO , and wild type (WT) HEK293T cells (C). Echo 6 infection in CD55 KO (G), with the above three cell lines in (C) as controls. TCID 50 was calculated at 48 h post infection (h.p.i). “+” and “-” indicate the presence or absence of FcRn or CD55 in the KO cell lines. (D and E) Echo 6 infection in FCGRT KO cells complemented with lentivirus expressing FCGRT (D) and B2M KO cells complemented with lentivirus expressing β2 m (E). KO cell lines with lentivirus expressing GFP was used as a control. Cells were infected with Echo 6 (MOI 0.01), and supernatants were harvested for virus titration at 24 h.p.i. (F) Light microscopy images showing CPE of FCGRT KO , B2M KO , CD55 KO , and WT HEK293T cells at 24 h.p.i with Echo 6 (MOI 1). Immunofluorescence images of KO and WT cells infected by Echo 6 (MOI 10) for 8 h and then stained by anti-Echo 6 antibodies. The scale bar represents 50 μm. (H) Growth curve of Echo 6 in FCGRT KO , B2M KO , CD55 KO , and WT HEK293T cells. (I) Echo 6 was inoculated onto non-susceptible CHO (left) and BHK (right) cells with ectopic expression of human FcRn (FCGRT+β2 m), CD55, or both. The data depict means with SEM. TCID 50 values were calculated by the Reed-Muench method. Experiments were repeated three times. # stands for undetected; ∗∗∗∗ p

    Article Snippet: Plasmid expressing human CD55 was transfected into HEK293T cells for CD55 expression.

    Techniques: CRISPR, Functional Assay, Infection, Expressing, Titration, Light Microscopy, Immunofluorescence, Staining, Endpoint Dilution Assay

    FcRn and CD55 Soluble Protein Expression and Purification, Related to Figure 3 (A) FcRn soluble protein was expressed by co-transfection of constructs encoding the FCGRT extracellular domain and B2M in HEK293T cells. The protein was eluted as a single peak from gel filtration on Superdex 200 column (GE Healthcare), and two bands showing FCGRT and B2M were evident by SDS-PAGE. (B) Gel filtration and SDS-PAGE of CD55 soluble protein purification.

    Journal: Cell

    Article Title: Human Neonatal Fc Receptor Is the Cellular Uncoating Receptor for Enterovirus B

    doi: 10.1016/j.cell.2019.04.035

    Figure Lengend Snippet: FcRn and CD55 Soluble Protein Expression and Purification, Related to Figure 3 (A) FcRn soluble protein was expressed by co-transfection of constructs encoding the FCGRT extracellular domain and B2M in HEK293T cells. The protein was eluted as a single peak from gel filtration on Superdex 200 column (GE Healthcare), and two bands showing FCGRT and B2M were evident by SDS-PAGE. (B) Gel filtration and SDS-PAGE of CD55 soluble protein purification.

    Article Snippet: Plasmid expressing human CD55 was transfected into HEK293T cells for CD55 expression.

    Techniques: Expressing, Purification, Cotransfection, Construct, Filtration, SDS Page, Protein Purification