Journal: Microbiology Spectrum

Article Title: Plasminogen deficiency reduces disease severity and immune responses in enterovirus A71-infected mice

doi: 10.1128/spectrum.03311-24

Figure Lengend Snippet: Anti-plasminogen antibody blocks the binding of EV-A71 to RD cells. ( A ) RD cells were treated with different concentrations of antibodies, including anti-SACRB2, anti-NCL, anti-PLG, and anti-SELS antibodies, for 1 h, followed by infection with EV-A71 for 3 h at 4°C. The bound virus was detected by flow cytometry with anti-EV-A71 antibody and FITC-conjugated second antibody. Preincubation of antibodies significantly reduced the binding of EV-A71 to RD cells in a dose-dependent manner. ( B ) PLG-virus preincubation facilitates EV-A71 binding to RD cells. ( C ) Knockdown of PLG attenuates EV-A71 binding to RD cells. ( D ) Virus-protein interaction detected by ELISA assay. Purified EV-A71 and BSA were diluted from 1 pg/50 µL to 10 ng/50 µL. The 100 pg/100 µL of PLG was added to each well. EV-A71 directly interacts with PLG. ( E ) The plates were coated with 1 ng/well of PLG. The purified EV-A71 was diluted in twofold serial dilution. The virus-protein interaction was detected by an SPR assay reader (EnSpire). ( F ) The virus-protein interaction was detected by the SPR assay reader (BIACore). The data were expressed as the mean ± SD of each group ( n ≥ 3). * P < 0.05.

Article Snippet: To evaluate whether the anti-PLG antibody influences the attachment of EV-A71 to host cells, RD cells were treated with or without specific antibodies, including anti-SCARB2 ( B97306 , Sigma), anti-NCL (ab22758, Abcam), anti-PLG (ab77811, Abcam), and anti-SELS (SAB2102105, Merck) antibodies for 1 h at 4 o C, and then incubated with EV-A71 for 3 h at 4 o C. The bound virus was detected by flow cytometry using an anti-EV-A71 antibody (MAB979, Millipore) and FITC-conjugated second antibody ( C04025 , Croyez).

Techniques: Binding Assay, Infection, Virus, Flow Cytometry, Knockdown, Enzyme-linked Immunosorbent Assay, Purification, Serial Dilution, SPR Assay

Journal: Microbiology Spectrum

Article Title: Plasminogen deficiency reduces disease severity and immune responses in enterovirus A71-infected mice

doi: 10.1128/spectrum.03311-24

Figure Lengend Snippet: The clinical scores, survival rates, and tissue viral loads of PLG-KO and WT mice in EV-A71 infection. Seven-day-old WT and PLG-KO mice were infected with the 5 × 10 4 pfu/mouse of EV71 strain. The clinical score ( A ) and the survival rates ( B ) of the WT and PLG-KO mice were measured for 2 weeks. PLG-KO mice showed lower clinical scores and mortality rates than WT mice. Clinical scores: 0, healthy; 1, reduced motility; 2, limb weakness; 3, limb paralysis; 4, moribund; and 5, death. Seven-day-old WT and PLG-KO mice were infected with 5 × 10 4 pfu/mouse of EV-A71 MP4 strain to evaluate the viral loads in the brain stem, spinal cord, and skeletal muscle. The WT ( n = 5) and PLG-KO mice ( n = 5) were sacrificed 3 days ( C ) and 6 days ( D ) post-infection. The virus titers were determined by plaque assay. The mean of virus titers (log10 pfu/g) in WT mice was red, and in PLG-KO mice was blue. * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: To evaluate whether the anti-PLG antibody influences the attachment of EV-A71 to host cells, RD cells were treated with or without specific antibodies, including anti-SCARB2 ( B97306 , Sigma), anti-NCL (ab22758, Abcam), anti-PLG (ab77811, Abcam), and anti-SELS (SAB2102105, Merck) antibodies for 1 h at 4 o C, and then incubated with EV-A71 for 3 h at 4 o C. The bound virus was detected by flow cytometry using an anti-EV-A71 antibody (MAB979, Millipore) and FITC-conjugated second antibody ( C04025 , Croyez).

Techniques: Infection, Virus, Plaque Assay

Journal: Microbiology Spectrum

Article Title: Plasminogen deficiency reduces disease severity and immune responses in enterovirus A71-infected mice

doi: 10.1128/spectrum.03311-24

Figure Lengend Snippet: PLG-KO decreases the disease progression of EV-A71-associated neuropathy. Seven-day-old WT and PLG-KO mice were infected with 5 × 10 4 pfu/mouse EV-A71 intraperitoneally. Mice were sacrificed on days 2 ( A ) and 6 ( B ). Paraffin-embedded skeletal muscle, spinal cord, and brain stem sections were examined with H&E stain at 200× magnifications. WT mice show cell neuropathy, such as proliferation, tissue damage, and lymphocytic infiltration in their nervous system at day 6 post-infection (arrow indicated). Negative control (mock): without virus infection.

Article Snippet: To evaluate whether the anti-PLG antibody influences the attachment of EV-A71 to host cells, RD cells were treated with or without specific antibodies, including anti-SCARB2 ( B97306 , Sigma), anti-NCL (ab22758, Abcam), anti-PLG (ab77811, Abcam), and anti-SELS (SAB2102105, Merck) antibodies for 1 h at 4 o C, and then incubated with EV-A71 for 3 h at 4 o C. The bound virus was detected by flow cytometry using an anti-EV-A71 antibody (MAB979, Millipore) and FITC-conjugated second antibody ( C04025 , Croyez).

Techniques: Biomarker Discovery, Infection, Staining, Negative Control, Virus

Journal: Microbiology Spectrum

Article Title: Plasminogen deficiency reduces disease severity and immune responses in enterovirus A71-infected mice

doi: 10.1128/spectrum.03311-24

Figure Lengend Snippet: PLG-KO decreases EV-A71 VP1 protein expression in tissues. Seven-day-old WT and PLG-KO mice were infected with 5 × 10 4 pfu/mouse EV-A71 intraperitoneally. Mice were sacrificed on day 6. Paraffin-embedded skeletal muscle, spinal cord, and brain stem sections were examined with IHC stain at 200× magnifications. IHC staining detected the most vital expression of viral VP1 protein in skeletal muscle, spinal cord, and brain stem in the WT mice (arrow indicated). Negative control (mock): without virus infection.

Article Snippet: To evaluate whether the anti-PLG antibody influences the attachment of EV-A71 to host cells, RD cells were treated with or without specific antibodies, including anti-SCARB2 ( B97306 , Sigma), anti-NCL (ab22758, Abcam), anti-PLG (ab77811, Abcam), and anti-SELS (SAB2102105, Merck) antibodies for 1 h at 4 o C, and then incubated with EV-A71 for 3 h at 4 o C. The bound virus was detected by flow cytometry using an anti-EV-A71 antibody (MAB979, Millipore) and FITC-conjugated second antibody ( C04025 , Croyez).

Techniques: Expressing, Infection, Staining, Immunohistochemistry, Negative Control, Virus

Journal: Microbiology Spectrum

Article Title: Plasminogen deficiency reduces disease severity and immune responses in enterovirus A71-infected mice

doi: 10.1128/spectrum.03311-24

Figure Lengend Snippet: Cytokine expression in the serum of WT and PLG-KO mice. Seven-day-old WT (white blocks) and PLG-KO (black blocks) mice were infected with 5 × 10 4 pfu/mouse EV-A71 intraperitoneally ( n = 5). Mice were sacrificed at 2, 4, and 6 days post-infection, and serum/spinal cord samples were collected. The expression of cytokines was evaluated. Cytokine concentrations were presented as pg/mL of the specimen. ( A ) IL-1β, ( B ) IL-6, ( C ) MCP-1, ( D ) IL-10, ( E ) IFN-γ, and ( F ) MCP-1 in the spinal cord. The nonparametric Mann-Whitney U -test determined each group’s statistical significance, the mean ± SD. * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: To evaluate whether the anti-PLG antibody influences the attachment of EV-A71 to host cells, RD cells were treated with or without specific antibodies, including anti-SCARB2 ( B97306 , Sigma), anti-NCL (ab22758, Abcam), anti-PLG (ab77811, Abcam), and anti-SELS (SAB2102105, Merck) antibodies for 1 h at 4 o C, and then incubated with EV-A71 for 3 h at 4 o C. The bound virus was detected by flow cytometry using an anti-EV-A71 antibody (MAB979, Millipore) and FITC-conjugated second antibody ( C04025 , Croyez).

Techniques: Expressing, Infection, MANN-WHITNEY

Journal: Avicenna Journal of Medical Biotechnology

Article Title: The Role of Disulfide Bridges in the Interaction of E. coli -Derived Recombinant SCARB2 and EV-A71’s Capsid

doi: 10.18502/ajmb.v17i1.17675

Figure Lengend Snippet: Expression of recombinant SCARB2 determined by SDS-PAGE with Coomassie brilliant blue staining (A, C) and confirmed by Western blot with anti-SCARB2-HRP (B, D) in E. coli BL21 (DE3) (A, B) and E. coli SHuffle ® T7 Express (C, D). M, protein maker (A, C) and pre-stained protein marker (B, D); 1, E. coli /pET-22b (+IPTG); 2, E. coli /pET-SCARB2 (−IPTG); 3–5, E. coli /pETSCARB2 (+IPTG); 3, total phase; 4, soluble phase; 5, insoluble phase.

Article Snippet: To assess protein expression, Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (using Tris-Glycine buffer with 12.5% gel in Bio-Rad electrophoresis systems) and Coomassie Brilliant Blue staining were conducted, followed by Western blot (using Cytiva blotting systems) with anti-SCARB2-Horseradish Peroxidase (HRP) antibody (Santa Cruz).

Techniques: Expressing, Recombinant, SDS Page, Staining, Western Blot, Marker

Journal: Avicenna Journal of Medical Biotechnology

Article Title: The Role of Disulfide Bridges in the Interaction of E. coli -Derived Recombinant SCARB2 and EV-A71’s Capsid

doi: 10.18502/ajmb.v17i1.17675

Figure Lengend Snippet: Refolding of SCARB2 inclusion bodies obtained from E. coli BL21(DE3)/pET-SCARB2 by rapid dilution method determined by SDS-PAGE with Silver staining (A) and verified by Western blot with anti-SCARB2-HRP (B). M, protein maker (A) and pre-stained protein marker (B); 1, inclusion bodies; 2, precipitated phase after dissolving; 3, supernatant phase after dissolving; 4, precipitated phase after refolding; 5, supernatant phase after refolding; 6, precipitated phase after dialysing; 7, supernatant phase after dialysing.

Article Snippet: To assess protein expression, Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (using Tris-Glycine buffer with 12.5% gel in Bio-Rad electrophoresis systems) and Coomassie Brilliant Blue staining were conducted, followed by Western blot (using Cytiva blotting systems) with anti-SCARB2-Horseradish Peroxidase (HRP) antibody (Santa Cruz).

Techniques: SDS Page, Silver Staining, Western Blot, Staining, Marker

Journal: Avicenna Journal of Medical Biotechnology

Article Title: The Role of Disulfide Bridges in the Interaction of E. coli -Derived Recombinant SCARB2 and EV-A71’s Capsid

doi: 10.18502/ajmb.v17i1.17675

Figure Lengend Snippet: Recovery efficiencies of the refolding processes

Article Snippet: To assess protein expression, Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (using Tris-Glycine buffer with 12.5% gel in Bio-Rad electrophoresis systems) and Coomassie Brilliant Blue staining were conducted, followed by Western blot (using Cytiva blotting systems) with anti-SCARB2-Horseradish Peroxidase (HRP) antibody (Santa Cruz).

Techniques:

Journal: Avicenna Journal of Medical Biotechnology

Article Title: The Role of Disulfide Bridges in the Interaction of E. coli -Derived Recombinant SCARB2 and EV-A71’s Capsid

doi: 10.18502/ajmb.v17i1.17675

Figure Lengend Snippet: Refolding of SCARB2 inclusion bodies from E. coli SHuffle ® T7 Express/pET-SCARB2 by dialysis method determined by SDS-PAGE with Silver staining (A) and verified by Western blot with anti-SCARB2-HRP (B). M, protein maker (A) and pre-stained protein marker (B); 1, inclusion bodies; 2, precipitated phase after dissolving; 3, supernatant phase after dissolving; 4, precipitated phase after dialysing; 5, supernatant phase after dialysing.

Article Snippet: To assess protein expression, Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (using Tris-Glycine buffer with 12.5% gel in Bio-Rad electrophoresis systems) and Coomassie Brilliant Blue staining were conducted, followed by Western blot (using Cytiva blotting systems) with anti-SCARB2-Horseradish Peroxidase (HRP) antibody (Santa Cruz).

Techniques: SDS Page, Silver Staining, Western Blot, Staining, Marker

Journal: Avicenna Journal of Medical Biotechnology

Article Title: The Role of Disulfide Bridges in the Interaction of E. coli -Derived Recombinant SCARB2 and EV-A71’s Capsid

doi: 10.18502/ajmb.v17i1.17675

Figure Lengend Snippet: Confirmation of disulfide bridges in recombinant SCARB2 after refolding. M, protein maker; 1, SCARB2/ E. coli BL21(DE3) (+DTT); 2, SCARB2/ E. coli SHuffle ® T7 Express (+DTT); 3, SCARB2/ E. coli BL21(DE3) (−DTT); 4, SCARB2/ E. coli SHuffle ® T7 Express (−DTT).

Article Snippet: To assess protein expression, Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (using Tris-Glycine buffer with 12.5% gel in Bio-Rad electrophoresis systems) and Coomassie Brilliant Blue staining were conducted, followed by Western blot (using Cytiva blotting systems) with anti-SCARB2-Horseradish Peroxidase (HRP) antibody (Santa Cruz).

Techniques: Recombinant

Journal: Avicenna Journal of Medical Biotechnology

Article Title: The Role of Disulfide Bridges in the Interaction of E. coli -Derived Recombinant SCARB2 and EV-A71’s Capsid

doi: 10.18502/ajmb.v17i1.17675

Figure Lengend Snippet: Evaluation of the interaction of recombinant SCARB2/ E. coli BL21(DE3) (A), and SCARB2/ E. coli SHuffle ® T7 Express (B) with EVA71’s capsid by indirect ELISA at different pHs.

Article Snippet: To assess protein expression, Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (using Tris-Glycine buffer with 12.5% gel in Bio-Rad electrophoresis systems) and Coomassie Brilliant Blue staining were conducted, followed by Western blot (using Cytiva blotting systems) with anti-SCARB2-Horseradish Peroxidase (HRP) antibody (Santa Cruz).

Techniques: Recombinant, Indirect ELISA

Journal: PLOS Pathogens

Article Title: Enterovirus A71 does not meet the uncoating receptor SCARB2 at the cell surface

doi: 10.1371/journal.ppat.1012022

Figure Lengend Snippet: (A) The previous model suggested by Yamayoshi et al. [ , ]. SCARB2 is highly expressed on the cell surface. Surface SCARB2 binds to EV-A71 at the south rim of the canon and initiates viral entry in endosomal vesicles. SCARB2 induces uncoating when the vesicle lumen becomes acidic. (B) Our new model. SCARB2 is absent from the cell surface. A non-SCARB2 receptor mediates EV-A71 attachment, most likely interacting with the viral five-fold vertex [ , ]. Virus is internalized and transported through the endosomal system, meeting SCARB2 within late endosomes or lysosomes, where uncoating occurs.

Article Snippet: To address this hypothesis, we employed two anti-SCARB2 Abs: first, a goat polyclonal Ab (pAb) (R&D systems, Cat# AF1966) used in the previous study to detect SCARB2 on the surface of RD cells [ ]; and second, a rabbit monoclonal Ab (mAb) (clone 12H5L1, Invitrogen, Cat # 702770).

Techniques: Virus

Journal: PLOS Pathogens

Article Title: Enterovirus A71 does not meet the uncoating receptor SCARB2 at the cell surface

doi: 10.1371/journal.ppat.1012022

Figure Lengend Snippet: (A) Flow cytometric analysis of cell-surface SCARB2 using goat pAb and rabbit mAb (clone 12H5L1). To confirm both Abs are applicable to flow cytometry, 293T cells overexpressing SCARB2 or mutant SCARB2 with the three amino acid substitutions to enhance cell-surface expression (SCARB2/QQG) and Jurkat cells were stained in parallel. The solid line and the shaded area represent staining with anti-SCARB2 Ab and control IgG, respectively, followed by Alexa Fluor 488-tagged secondary Ab. The figure is representative of three independent experiments. (B) A large amount of overexpressed SCARB2 existed inside the 293T cells. Western blotting analysis with anti-SCARB2 mAb (clone 12H5L1). Recombinant SCARB2-Fc (5 ng) was loaded as a positive control. RD and RD-SCARB2-KO (clone No.3) cells were loaded as positive and negative controls, respectively. The figure is representative of three independent experiments.

Article Snippet: To address this hypothesis, we employed two anti-SCARB2 Abs: first, a goat polyclonal Ab (pAb) (R&D systems, Cat# AF1966) used in the previous study to detect SCARB2 on the surface of RD cells [ ]; and second, a rabbit monoclonal Ab (mAb) (clone 12H5L1, Invitrogen, Cat # 702770).

Techniques: Flow Cytometry, Mutagenesis, Expressing, Staining, Western Blot, Recombinant, Positive Control

Journal: PLOS Pathogens

Article Title: Enterovirus A71 does not meet the uncoating receptor SCARB2 at the cell surface

doi: 10.1371/journal.ppat.1012022

Figure Lengend Snippet: Jurkat cells were pre-treated with anti-PSGL-1 mAb (10 μg ml -1 ), anti-SCARB2 pAb (50 μg ml -1 ), or control Abs on ice for 1 h, followed by infection with EV-A71 at 1 CCID 50 per cell on ice for 1 h. Cells were then washed (0 h), cultured without Abs, and harvested at 5 days (5 d) post-infection. Results are indicated as the mean and s.e. for triplicate samples.

Article Snippet: To address this hypothesis, we employed two anti-SCARB2 Abs: first, a goat polyclonal Ab (pAb) (R&D systems, Cat# AF1966) used in the previous study to detect SCARB2 on the surface of RD cells [ ]; and second, a rabbit monoclonal Ab (mAb) (clone 12H5L1, Invitrogen, Cat # 702770).

Techniques: Infection, Cell Culture

Journal: PLOS Pathogens

Article Title: Enterovirus A71 does not meet the uncoating receptor SCARB2 at the cell surface

doi: 10.1371/journal.ppat.1012022

Figure Lengend Snippet: PSGL-1 or SCARB2 were knocked out by CRISPR/Cas9 in Jurkat cells, and two each of clones were established. (A) Cells were infected with EV-A71 at 1 CCID 50 per cell on ice for 1 h, then washed (0 h), cultured, and harvested at 3 days (3 d) post-infection. (B) Cells were infected with EV-A71-EGFP at 10 CCID 50 per cell and cultured for 18 h. Infected cells were identified by flow cytometry to detect EGFP expression. Results are indicated as the mean and s.e. for triplicate samples.

Article Snippet: To address this hypothesis, we employed two anti-SCARB2 Abs: first, a goat polyclonal Ab (pAb) (R&D systems, Cat# AF1966) used in the previous study to detect SCARB2 on the surface of RD cells [ ]; and second, a rabbit monoclonal Ab (mAb) (clone 12H5L1, Invitrogen, Cat # 702770).

Techniques: CRISPR, Clone Assay, Infection, Cell Culture, Flow Cytometry, Expressing

Journal: PLOS Pathogens

Article Title: Enterovirus A71 does not meet the uncoating receptor SCARB2 at the cell surface

doi: 10.1371/journal.ppat.1012022

Figure Lengend Snippet: RD, HeLa, HEp-2, 293T, and Hep G2 were obtained from the ATCC specifically for this study and used after limited passage. (A) Western blotting analysis by anti-SCARB2 pAb (left) and mAb (right, clone 12H5L1). Recombinant SCARB2-Fc (1 ng for pAb, 5 ng for mAb) was loaded as a positive control. RD-SCARB2-KO clones were loaded as negative controls. The figure is representative of three independent experiments. (B) Flow cytometric analysis by anti-SCARB2 pAb (top panels) and mAb (bottom panels), followed by Alexa Fluor 488-tagged secondary Ab. The solid line and the shaded area represent staining with anti-SCARB2 Ab and control Ab, respectively. Note that the solid line and the border of the shaded area are almost completely overlapped, indicating the absence of SCARB2 on the cell surface. Representative results with the following passage numbers after receiving from the ATCC; for pAb: RD, 3; HeLa, 3; HEp-2, 3; 293T, 3; Hep G2, 5; for mAb: RD, 13; HeLa, 5; HEp-2, 4; 293T, 4; Hep G2, 8. As a positive control of SCARB2 staining, cells expressing surface SCARB2 were always stained and analyzed in parallel. The figure is representative of three independent experiments. (C) EGFP expression in cells infected with EV-A71-EGFP. Cells were infected with EV-A71-EGFP at 10 CCID 50 per cell and cultured for 18 h. Then EGFP expression was measured by flow cytometry. The EGFP-negative cells are not infected. The majority of EGFP-dim cells were infected early in the incubation period, and are dying and losing EGFP expression; some may have just been infected and are starting to express EGFP. The EGFP-bright cells were infected late in the incubation period are actively producing EGFP. The number indicates the percentage of EGFP-positive cells (mean and s.e. for three independent experiments).

Article Snippet: To address this hypothesis, we employed two anti-SCARB2 Abs: first, a goat polyclonal Ab (pAb) (R&D systems, Cat# AF1966) used in the previous study to detect SCARB2 on the surface of RD cells [ ]; and second, a rabbit monoclonal Ab (mAb) (clone 12H5L1, Invitrogen, Cat # 702770).

Techniques: Western Blot, Recombinant, Positive Control, Clone Assay, Staining, Expressing, Infection, Cell Culture, Flow Cytometry, Incubation

Journal: PLOS Pathogens

Article Title: Enterovirus A71 does not meet the uncoating receptor SCARB2 at the cell surface

doi: 10.1371/journal.ppat.1012022

Figure Lengend Snippet: (A) Cells were stained with anti-SCARB2 mAb (clone 22H6L14) followed by Alexa Fluor 488-tagged secondary Ab and WGA conjugated with Alexa Fluor 633 without permeabilization. WGA was used to visualize the plasma membrane. Then the cells were fixed and observed under a confocal microscope. RD-SCARB2-KO cells (clone No.3) and 293T cells transfected with a control plasmid were used as negative controls. 293T cells expressing SCARB2/QQG on the cell surface were used as positive control. The figure is representative of three independent experiments. (B) RD and RD-SCARB2-KO (clone No.3) cells were stained with WGA, fixed, permeabilized, and stained with anti-SCARB2 mAb (clone 22H6L14) followed by Alexa Fluor 488-tagged secondary Ab. The figure is representative of three independent experiments. Scale bars, 10 μm.

Article Snippet: To address this hypothesis, we employed two anti-SCARB2 Abs: first, a goat polyclonal Ab (pAb) (R&D systems, Cat# AF1966) used in the previous study to detect SCARB2 on the surface of RD cells [ ]; and second, a rabbit monoclonal Ab (mAb) (clone 12H5L1, Invitrogen, Cat # 702770).

Techniques: Staining, Membrane, Microscopy, Transfection, Plasmid Preparation, Expressing, Positive Control

Journal: PLOS Pathogens

Article Title: Enterovirus A71 does not meet the uncoating receptor SCARB2 at the cell surface

doi: 10.1371/journal.ppat.1012022

Figure Lengend Snippet: RD cells were fixed, permeabilized, and stained with anti-SCARB2 mAb (clone 22H6L14) and mAb against either EEA1 (early endosome), CD63 (late endosome), or LAMP-1 (lysosome), followed by Alexa Fluor-tagged secondary Ab. Then the cells were observed under a confocal microscope. The figure is representative of three independent experiments. In each experiment, five pairs of images were analyzed. The graph shows colocalization between the markers and SCARB2 expressed as Pearson’s correlation coefficient. The vertical line indicates the mean value. Scale bars, 10 μm.

Article Snippet: To address this hypothesis, we employed two anti-SCARB2 Abs: first, a goat polyclonal Ab (pAb) (R&D systems, Cat# AF1966) used in the previous study to detect SCARB2 on the surface of RD cells [ ]; and second, a rabbit monoclonal Ab (mAb) (clone 12H5L1, Invitrogen, Cat # 702770).

Techniques: Staining, Microscopy

Journal: PLOS Pathogens

Article Title: Enterovirus A71 does not meet the uncoating receptor SCARB2 at the cell surface

doi: 10.1371/journal.ppat.1012022

Figure Lengend Snippet: (A) EV-A71 binding to RD cells in the presence of anti-SCARB2 pAb. RD cells pretreated with the anti-SCARB2 pAb (50 μg ml -1 ) were reacted with EV-A71 (4 × 10 8 genome copies) on ice for 30 min. Then the cells were washed, and cellular and viral nucleotides were extracted. EV-A71 bound to the cell were analyzed by real-time RT-PCR by ΔΔCt method using ATP5F1 mRNA as an endogenous control. As a technical control of detection of reduced copy number, quarter (1 × 10 8 genome copies) and half (2 × 10 8 genome copies) amount of EV-A71 was tested in parallel. The relative virus binding of RD cells reacted with 4 × 10 8 genome copies of EV-A71 without Ab was expressed as 1. (B) EGFP expression in cells infected with EV-A71-EGFP in the presence of anti-SCARB2 pAb at 18 h post-infection. (C) EV-A71 binding to RD and RD-SCARB2-KO clones. EV-A71 bound to the cell were analyzed as in (A). The relative virus binding of RD cells reacted with 4 × 10 8 genome copies of EV-A71 was expressed as 1. (D) Replication kinetics of EV-A71 in RD and RD-SCARB2-KO clones. Statistical significance was measured for each time point. Results are indicated as the mean and s.e. for three independent experiments (A, B, C) or triplicate analyses (D). Asterisks indicate P < 0.0001.

Article Snippet: To address this hypothesis, we employed two anti-SCARB2 Abs: first, a goat polyclonal Ab (pAb) (R&D systems, Cat# AF1966) used in the previous study to detect SCARB2 on the surface of RD cells [ ]; and second, a rabbit monoclonal Ab (mAb) (clone 12H5L1, Invitrogen, Cat # 702770).

Techniques: Binding Assay, Quantitative RT-PCR, Virus, Expressing, Infection, Clone Assay

Journal: PLOS Pathogens

Article Title: Enterovirus A71 does not meet the uncoating receptor SCARB2 at the cell surface

doi: 10.1371/journal.ppat.1012022

Figure Lengend Snippet: Human dermal fibroblasts (neonatal), intestinal fibroblasts, and tonsil endothelial cells were examined as cells presumed to be involved in the in vivo pathogenesis of EV-A71 infection. (A) Western blotting analysis with anti-SCARB2 mAb (clone 12H5L1). Recombinant SCARB2-Fc (5 ng) was loaded as a positive control. RD and RD-SCARB2-KO (clone No.3) cells were loaded as positive and negative controls, respectively. The figure is representative of three independent experiments. The graph displays the relative level of SCARB2 expression normalized by actin. The relative amount of SCARB2 in RD cells was expressed as 1. (B) Flow cytometric analysis by anti-SCARB2 pAb, followed by Alexa Fluor 488-tagged secondary Ab. The solid line and the shaded area represent staining with anti-SCARB2 pAb and control Ab, respectively. Note that the solid line and the border of the shaded area are almost completely overlapped, indicating the absence of SCARB2 on the cell surface. Representative results of cells passaged twice after receiving from the company. As a positive control of SCARB2 staining, cells expressing surface SCARB2 were always stained and analyzed in parallel. The figure is representative of three independent experiments. (C) EGFP expression in cells infected with EV-A71-EGFP in the presence of anti-SCARB2 pAb at 18 h post-infection. Cells with the following passage numbers after receiving from the company were used; dermal fibroblasts, 4; intestinal fibroblasts, 4; tonsil endothelial cells, 2. Results are indicated as the mean and s.e. for three independent experiments (A, C).

Article Snippet: To address this hypothesis, we employed two anti-SCARB2 Abs: first, a goat polyclonal Ab (pAb) (R&D systems, Cat# AF1966) used in the previous study to detect SCARB2 on the surface of RD cells [ ]; and second, a rabbit monoclonal Ab (mAb) (clone 12H5L1, Invitrogen, Cat # 702770).

Techniques: In Vivo, Infection, Western Blot, Recombinant, Positive Control, Expressing, Staining