Journal: eLife

Article Title: LRG1 is an adipokine that promotes insulin sensitivity and suppresses inflammation

doi: 10.7554/eLife.81559

Figure Lengend Snippet: ( A ) Search strategy for identification of novel adipokines with a potential role in whole-body metabolism. ( B ) Relative Lrg1 mRNA levels across a panel of tissues from 8-week-old male B6 mice. Visc, visceral fat depots; SubQ, subcutaneous fat depots. n=4–5 biological replicates per group. ( C ) Relative Lrg1 mRNA levels of indicated adipose tissues fractionated into mature adipocytes and SVF. ****p<0.0001 from Šídák post hoc test following two-way ANOVA. n=4 biological replicates per group. ( D ) Relative Lrg1 mRNA levels during in vitro differentiation of primary SVF into adipocytes. n=3 technical replicates per group. ( E ) Western blot of LRG1 and CFD in CM before (day 0) and after (day 7) in vitro adipogenic differentiation, in technical replicates. ( F ) Serum western blot of LRG1 from male B6 mice on standard chow or HFD for indicated weeks. HFD was started at 6 weeks of age. Each lane represents a biological replicate. ( G ) Relative Lrg1 mRNA levels of indicated tissues from mice on standard chow or HFD for 15 weeks. HFD was started at 6 weeks of age. ***p<0.001 from two-sided Welch’s t -test. n=6 biological replicates per group. ( H ) Relative Lrg1 mRNA levels of primary SubQ adipocytes treated with recombinant TNFα and 850 nM insulin for 6 hr. **p<0.01, ***p<0.001 from Dunnett post hoc test following one-way ANOVA. n=3 technical replicates per group. ( I ) Western blot of LRG1 in CM of primary SubQ adipocytes treated with PBS or 100 ng/mL recombinant TNFα without insulin for 24 hr. Each lane represents a technical replicate. Data are presented as mean ± SEM. Figure 3—source data 1. Uncropped gel and western blot images in and . Figure 3—source data 2. Raw gel and western blot images in and .

Article Snippet: Peptide, recombinant protein , Recombinant human TNFα , R and D Systems , Cat# 210-TA-020/CF , .

Techniques: In Vitro, Western Blot, Recombinant

Journal: eLife

Article Title: LRG1 is an adipokine that promotes insulin sensitivity and suppresses inflammation

doi: 10.7554/eLife.81559

Figure Lengend Snippet: ( A ) α-FLAG co-IP of C-terminally FLAG-tagged LRG1 and Cyt c from the serum of AAV-transduced db/db mice at 7 weeks of age. ( B ) Western blot of Cyt c in the serum of db/db or littermate m/m mice at 7 or 10 weeks of age. ( C ) Western blot of Cyt c in the serum of B6 mice on standard chow or HFD for indicated weeks. HFD was started at 6 weeks of age. Developed from the same membrane in . ( D ) Western blot of Cyt c in the CM of primary SubQ adipocytes under conditions promoting cell death. SubQ cells were treated with 1 µM staurosporine (STS) or DMSO for 24 hr (top); placed in normoxic or hypoxic chambers for 24 hr (middle); or treated with 100 ng/mL recombinant TNFα or PBS without insulin for 24 hr. Developed from the same membrane in (bottom). ( E ) Western blot of Cyt c in the insterstitial fluid (ISF) of eWAT and iWAT from B6 mice on standard chow or HFD for indicated weeks. ( F ) Relative mRNA levels of a panel of pro-inflammatory genes in BMDMs in response to 30 µg/mL equine Cyt c or 10 µM TAK-242, a TLR4 inhibitor. BMDMs were treated with Cyt c or PBS for 6 hr following 1 hr pre-incubation with TAK-242 or DMSO. ****p<0.0001 from Tukey post hoc test results following two-way ANOVA. n=3 technical replicates per group. ( G ) Relative mRNA levels of a panel of pro-inflammatory genes in BMDMs in response to 20 µg/mL equine Cyt c and 50 µg/mL recombinant human LRG1. Indicated reagents or PBS were rotated for 1 hr at RT prior to BMDM treatment for 6 hr. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 from Tukey post hoc test results following two-way ANOVA. n=3–4 technical replicates per group. ( H ) Western blot of intact and cleaved Caspase-3 in eWAT of AAV-transduced db/db mice. ( I ) Western blot of LRG1 and Cyt c in the serum and ISF of db/db mice at 10 weeks of age. Vinculin was used to assess tissue leakage during sample preparation. ( J ) Schematic depicting proposed mechanism. Data are presented as mean ± SEM. Figure 7—source data 1. Labeled uncropped western blot images in and . Figure 7—source data 2. Raw western blot images in and .

Article Snippet: Peptide, recombinant protein , Recombinant human TNFα , R and D Systems , Cat# 210-TA-020/CF , .

Techniques: Co-Immunoprecipitation Assay, Western Blot, Membrane, Recombinant, Incubation, Sample Prep, Labeling

Journal: eLife

Article Title: LRG1 is an adipokine that promotes insulin sensitivity and suppresses inflammation

doi: 10.7554/eLife.81559

Figure Lengend Snippet:

Article Snippet: Peptide, recombinant protein , Recombinant human TNFα , R and D Systems , Cat# 210-TA-020/CF , .

Techniques: Produced, CRISPR, Electroporation, Plasmid Preparation, Western Blot, Magnetic Beads, Immunoprecipitation, Recombinant, Protein Enrichment, Sequencing

Journal: bioRxiv

Article Title: Methylation of KRAS at lysine 182 and 184 by SETD7 promotes KRAS degradation

doi: 10.1101/2021.02.10.429309

Figure Lengend Snippet: (A) Proteins from the co-immunoprecipitation (co-IP) assay using anti-HA magnetic beads in 293T with ectopic expression of 3×HA-tagged KRAS were separated by polyacrylamide gel electrophoresis and visualized using silver staining. (B) The workflow for identifying KRAS-interacting proteins was shown, including co-IP assay, sample preparation, LC-MS/MS analysis, protein quantification, and further computational analyses for differential proteins. (C) Volcano plot of proteins identified by LC-MS/MS analysis in HA-vector and 3×HA-KRAS groups. The ratio (KRAS/HA) and the mean of protein spectra counts (MeanSP) in two groups were calculated. The x axis shows the log2 ratio (KRAS/HA) and the y axis shows the log2 MeanSP. The proteins distributed outside the dark-red boundary line (y = log 2 2.5 / (x-1)) were labeled as significant differential proteins in KRAS group. TOP 10 differential proteins from each group were labeled. (D) Western blotting (WB) analysis of whole-cell lysates and immunoprecipitation (IP) of SETD7 in 293T cells transfected with the indicated plasmids. (E) Immunofluorescence staining of SETD7 (green) and KRAS (red) in 293T cells co-transfected with SETD7 and KRAS. DAPI was used to label the nuclei. Scale bar, 10 µm. (F and G) WB analysis of the co-IP assay using anti-SETD7 (F) or anti-RAS (G) antibodies in H358-SETD7-O/E stable cells. ( H ) WB analysis of the co-IP assay with anti-SETD7 antibody in non-small-cell lung cancer (NSCLC) cell line A549.

Article Snippet: The human non-small-cell lung cancer (NSCLC) cell lines A549, NCI-H358, NCI-H1437, and NCI-H522 were obtained from the American Type Culture Collection (ATCC) and cultured in RPMI1640 (Life Technologies) containing 10% FBS (PAN Seratech) and 100 U/mL penicillin-streptomycin (Life Technologies).

Techniques: Co-Immunoprecipitation Assay, Magnetic Beads, Expressing, Polyacrylamide Gel Electrophoresis, Silver Staining, Sample Prep, Liquid Chromatography with Mass Spectroscopy, Plasmid Preparation, Labeling, Western Blot, Immunoprecipitation, Transfection, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: Methylation of KRAS at lysine 182 and 184 by SETD7 promotes KRAS degradation

doi: 10.1101/2021.02.10.429309

Figure Lengend Snippet: (A) Two representative MS2 spectra of SETD7 peptides (116-143 and 324-344) from 3×HA-KRAS group. (B) Western blotting (WB) analysis of SETD7 expression in NSCLC cell lines from the American Type Culture Collection (ATCC). Staging information is shown above each cell line in red font.

Article Snippet: The human non-small-cell lung cancer (NSCLC) cell lines A549, NCI-H358, NCI-H1437, and NCI-H522 were obtained from the American Type Culture Collection (ATCC) and cultured in RPMI1640 (Life Technologies) containing 10% FBS (PAN Seratech) and 100 U/mL penicillin-streptomycin (Life Technologies).

Techniques: Western Blot, Expressing

Journal: bioRxiv

Article Title: Methylation of KRAS at lysine 182 and 184 by SETD7 promotes KRAS degradation

doi: 10.1101/2021.02.10.429309

Figure Lengend Snippet: (A-B) Cell proliferation (A) and two-dimensional (2-D) colony formation (B) of A549-vector and A549-SETD7-OE stable cells was measured separately. (C) WB analysis of SETD7 and RAS-related signaling pathways in A549 stable cells. (D-E) Cell proliferation (D), 2-D colony formation (E) of H358-control and two SETD7-KD stable cells were measured separately. (F-G) 3-D spheroid growth of H1437 (F) and H358 (G) controls and two SETD7-KD stable cells was measured separately. (H) WB analysis of SETD7 and RAS-related signaling pathways in H358 stable cells. * P <0.05; ** P <0.01; *** P <0.001.

Article Snippet: The human non-small-cell lung cancer (NSCLC) cell lines A549, NCI-H358, NCI-H1437, and NCI-H522 were obtained from the American Type Culture Collection (ATCC) and cultured in RPMI1640 (Life Technologies) containing 10% FBS (PAN Seratech) and 100 U/mL penicillin-streptomycin (Life Technologies).

Techniques: Plasmid Preparation, Protein-Protein interactions, Control

Journal: bioRxiv

Article Title: Methylation of KRAS at lysine 182 and 184 by SETD7 promotes KRAS degradation

doi: 10.1101/2021.02.10.429309

Figure Lengend Snippet: (A) Measurement of volumes of xenograft tumors derived from A549-vector or A549-SETD7-O/E stable cells (n = 8). (B) A photograph of the xenograft tumors and the tumor weights, 7 weeks after cell injection, are shown (upper and lower panels, respectively). (C) Representative images of immunohistochemical (IHC) staining of SETD7, RAS, and Ki67 in xenograft tumor tissues. Scale bar, 100 μm. (D) Measurement of volumes of xenograft tumors derived from H1437-control and two SETD7-KD stable cells (n = 10). (E) A photograph of the xenograft tumors and the tumor weights, 4 weeks after cell injection, are shown (upper and lower panels, respectively; scale bar, 10 mm). (F) Representative images of IHC staining of SETD7, RAS, and Ki67 in xenograft tumor tissues of each group. Scale bar, 100 μm. * P <0.05; ** P <0.01; *** P <0.001.

Article Snippet: The human non-small-cell lung cancer (NSCLC) cell lines A549, NCI-H358, NCI-H1437, and NCI-H522 were obtained from the American Type Culture Collection (ATCC) and cultured in RPMI1640 (Life Technologies) containing 10% FBS (PAN Seratech) and 100 U/mL penicillin-streptomycin (Life Technologies).

Techniques: Derivative Assay, Plasmid Preparation, Injection, Immunohistochemical staining, Immunohistochemistry, Control

Journal: bioRxiv

Article Title: Methylation of KRAS at lysine 182 and 184 by SETD7 promotes KRAS degradation

doi: 10.1101/2021.02.10.429309

Figure Lengend Snippet: (A) Tumor volumes of xenografts derived from A549 vector and SETD7-OE stable pools were measured 7 weeks after tumor cell injection. ** P <0.01. (B) Tumor volumes of xenografts derived from H1437 vector and two SETD7-KD stable pools were measured 4 weeks after tumor cell injection. *** P <0.001.

Article Snippet: The human non-small-cell lung cancer (NSCLC) cell lines A549, NCI-H358, NCI-H1437, and NCI-H522 were obtained from the American Type Culture Collection (ATCC) and cultured in RPMI1640 (Life Technologies) containing 10% FBS (PAN Seratech) and 100 U/mL penicillin-streptomycin (Life Technologies).

Techniques: Derivative Assay, Plasmid Preparation, Injection

Journal: bioRxiv

Article Title: Methylation of KRAS at lysine 182 and 184 by SETD7 promotes KRAS degradation

doi: 10.1101/2021.02.10.429309

Figure Lengend Snippet: (A-C) KRAS mRNA expression was detected by qPCR in A549 (A) and H358 (B) stable cells with SETD7 overexpression, or H1437 (C) stable cells with SETD7 knockdown. (D) WB analysis of A549-vector and A549-SETD7-O/E stable cells with CHX (100 μg/mL) treatment, as indicated. The relative RAS protein level is labeled in red. (E) Cell proliferation of A549 vector, SETD7 WT , and SETD7 H297A stable cells was measured using a CCK-8 assay. (F-G) Two-dimensional (2-D) colony formation (F) and 3-D spheroid growth (G) of A549 vector, SETD7 WT , and SETD7 H297A stable cells were measured separately. The bar graphs represent the quantification of numbers of colonies on plates or spheroids in dishes with a cell-repellent surface, from three independent experiments. (H) WB analysis of SETD7 and RAS-related signaling pathways in A549 stable cells. * P <0.05, *** P <0.001. ns, not significant.

Article Snippet: The human non-small-cell lung cancer (NSCLC) cell lines A549, NCI-H358, NCI-H1437, and NCI-H522 were obtained from the American Type Culture Collection (ATCC) and cultured in RPMI1640 (Life Technologies) containing 10% FBS (PAN Seratech) and 100 U/mL penicillin-streptomycin (Life Technologies).

Techniques: Expressing, Over Expression, Knockdown, Plasmid Preparation, Labeling, CCK-8 Assay, Protein-Protein interactions

Journal: Cell

Article Title: Mapping Neutralizing and Immunodominant Sites on the SARS-CoV-2 Spike Receptor-Binding Domain by Structure-Guided High-Resolution Serology

doi: 10.1016/j.cell.2020.09.037

Figure Lengend Snippet: Kinetics of IgG Responses Specific for the SARS-CoV-2 RBD and Blocking RBD Attachment to ACE2 (A) Binding titers (ED50) of serum or plasma IgG to the SARS-CoV-2 RBD measured at two time points separated by an average time of 44 days in 368 subjects. T1, time of first blood draw; T2, time of second blood draw. (B) Variation of RBD-specific IgG binding titers from T1 to T2. (C) Kinetics of RBD- and N-specific IgG responses in serum or plasma from 24 convalescent individuals (red, hospitalized; blue, symptomatic non-hospitalized). The starting time point corresponds to the date of collection of the first sample. (D) Model predicted longitudinal decline of RBD- and N-specific IgG binding titers from 18 convalescent individuals with respect to the onset of symptoms from infection. Symbols, observations; shaded region, 90% prediction interval; line, median prediction. (E) Serum or plasma titers of Abs blocking RBD attachment to ACE2 (BD80) measured at T1 and T2. (F) Variation of RBD-specific IgG binding titers and titers of Abs blocking RBD attachment to ACE2 (BD80) from T1 to T2. (G) Avidity index of serum IgG binding to RBD (%) measured at T1 and T2. (H) Variation of avidity index of IgG binding to RBD (%) from T1 to T2.

Article Snippet: ACE2 Protein, Human , Sino Biological , Cat. No. 10108-H08H.

Techniques: Blocking Assay, Binding Assay, Infection

Journal: Cell

Article Title: Mapping Neutralizing and Immunodominant Sites on the SARS-CoV-2 Spike Receptor-Binding Domain by Structure-Guided High-Resolution Serology

doi: 10.1016/j.cell.2020.09.037

Figure Lengend Snippet: Analysis of the Specificity of IgG, IgA, and IgM Serum/Plasma Abs from a Panel of 647 Hospitalized, Symptomatic, and Asymptomatic SARS-CoV-2-Infected Individuals (A–C) Binding titers (ED50) of antigen-specific IgG (A), IgA (B), or IgM (C) were measured in plasma or sera from convalescent SARS-CoV-2 patients (47 hospitalized, 556 symptomatic, and 44 asymptomatic) and from pre-pandemic healthy donors (n = 32). A cut-off of 30 was determined based on signal of pre-pandemic samples and binding to uncoated ELISA plates. (D) Binding titers (ED50) of S- and N-specific IgGs measured in sera from symptomatic and asymptomatic SARS-CoV-2-infected individuals from the Ticino healthcare workers cohort (n = 459) categorized according to symptoms severity, as described in the methods. (E) IgG binding titers to SARS-CoV-2 RBD (left) and SARS-CoV-2 S pseudovirus neutralizing titers (ID80, center) before and after depletion of RBD-specific Abs from 21 SARS-CoV-2 immune plasma samples. The percentage of depletion of binding and neutralizing Abs (right) for each sample tested is shown on the right. (F) Ab-mediated inhibition of SARS-CoV-2 RBD binding to solid phase ACE2, as determined by ELISA. Shown is the reciprocal plasma or serum dilution that blocks 80% binding (BD80) of RBD to human ACE2. (G) Ab-mediated inhibition of SARS-CoV-2 RBD binding to solid phase ACE2 in the Ticino healthcare workers cohort determined as in (F). A cut-off of 10 was used to separate neutralizing from non-neutralizing titers. (H) Correlation analysis between levels of plasma/serum RBD-specific IgG (ED50) and the titers of Abs blocking RBD attachment to ACE2 (BD80). (I) Correlation analysis between plasma/serum neutralizing Ab titers (ID80) and the titers of Abs blocking RBD attachment to ACE2 (BD80).

Article Snippet: ACE2 Protein, Human , Sino Biological , Cat. No. 10108-H08H.

Techniques: Infection, Binding Assay, Enzyme-linked Immunosorbent Assay, Inhibition, Blocking Assay

Journal: Cell

Article Title: Mapping Neutralizing and Immunodominant Sites on the SARS-CoV-2 Spike Receptor-Binding Domain by Structure-Guided High-Resolution Serology

doi: 10.1016/j.cell.2020.09.037

Figure Lengend Snippet: Characteristics of the Six Probe mAbs Used for Structural and Epitope-Mapping Studies, Related to , , , , and (A) V(D)J usage, percentage identity to germline, number of somatic mutations, source and time interval between sample collection and mAb isolation, RBD site recognized and neutralization potency of the 6 mAbs. B mem, memory B cell; PC, plasma cells. (B) Binding of the 6 mAbs to the SARS-CoV-2 (up) or SARS-CoV (down) RBD analyzed by ELISA. (C) Competition matrix for binding of each of the six mAbs in presence of another mAb evaluated by biolayer interferometry. (D) mAb-mediated inhibition of RBD binding to ACE2 analyzed by ELISA. (E) mAb-mediated S 1 subunit shedding from cell-surface expressed SARS-CoV-2 S as determined by flow-cytometry. (F) Conservation of RBM and epitope residues in ∼74,000 SARS-CoV-2 sequences (GISAID, August 11 th , 2020). RBM and epitope residues are shown as gray bars. Black bars indicate variant prevalence for epitope residues with at least 2 variants. RBM residues were determined from PDB 6M0J using a 5.0 Å distance cutoff between RBD and ACE2 residues using MOE. (G) Western-blot analysis (top) of the prefusion-stabilized SARS-CoV-2 S ectodomain trimer in presence of S2A4, S304 or S2X35 Fab after incubation for the indicated amount of times. Red ponceau staining (bottom) of the SDS-PAGE gel used for carrying out the western blot confirming the presence of added Fabs when indicated. (H) Analysis of activation of FcγRIIIa (V158 allele) expressed on Jurkat cells by SARS-CoV-2 S stably transfected CHO cells incubated with mAbs. GRLR indicates an antibody Fc variant carrying mutations that abolish binding to FcγRs. (I) Analysis of activation of FcγRIIa (H131 allele), expressed on Jurkat cells by SARS-CoV-2 S stably transfected CHO cells incubated with mAbs. (J) Killing of SARS-CoV-2 S stably transfected CHO cells by mAbs in the presence of complement (CDC assay).

Article Snippet: ACE2 Protein, Human , Sino Biological , Cat. No. 10108-H08H.

Techniques: Isolation, Neutralization, Binding Assay, Enzyme-linked Immunosorbent Assay, Inhibition, Flow Cytometry, Variant Assay, Western Blot, Incubation, Staining, SDS Page, Activation Assay, Stable Transfection, Transfection, CDC Assay

Journal: Cell

Article Title: Mapping Neutralizing and Immunodominant Sites on the SARS-CoV-2 Spike Receptor-Binding Domain by Structure-Guided High-Resolution Serology

doi: 10.1016/j.cell.2020.09.037

Figure Lengend Snippet: The S2H13 mAb Inhibits SARS-CoV-2 by Blocking Attachment to ACE2 via Recognition of an Epitope Accessible in the Open and Closed S Conformations (A) SARS-CoV-2 S pseudovirus neutralization assay indicating an IC50 of 500 ng/mL. (B and C) Molecular surface representation of the SARS-CoV-2 S/S2H13 Fab complex structure with three RBDs closed shown in two orthogonal orientations. (D) Molecular surface representation of the SARS-CoV-2 S/S2H13 Fab complex structure with one RBD open. Each SARS-CoV-2 protomer is colored distinctly (cyan, pink, and gold), and N-linked glycans are rendered as dark blue surfaces. The S2H13 light and heavy chain variable domains are colored magenta and purple, respectively. (E) S2H13 recognizes a crevice formed by the SARS-CoV-2 RBM. Selected side chains at the interface are shown. (F) S2H13 and ACE2 (dark green) bind overlapping RBM epitope. The red star indicates steric clashes. (G) BLI binding competition between S2H13 and ACE2 for binding to the SARS-CoV-2 RBD. (H) Molecular surface representation of the SARS-CoV-2 RBD (gray) with the S2H13 epitope colored by residue conservation across SARS-CoV-2 isolates and SARS-CoV.

Article Snippet: ACE2 Protein, Human , Sino Biological , Cat. No. 10108-H08H.

Techniques: Blocking Assay, Neutralization, Binding Assay

Journal: Cell

Article Title: Mapping Neutralizing and Immunodominant Sites on the SARS-CoV-2 Spike Receptor-Binding Domain by Structure-Guided High-Resolution Serology

doi: 10.1016/j.cell.2020.09.037

Figure Lengend Snippet: The S2H14 mAb Inhibits SARS-CoV-2 by Blocking Attachment to the ACE2 Receptor (A) SARS-CoV-2 S pseudovirus neutralization assay indicating an IC50 of 900 ng/mL. (B and C) Molecular surface representation of the SARS-CoV-2 S/S2H14 Fab complex structure with two RBDs open and one RBD closed viewed along two orthogonal orientations. (D and E) Molecular surface representation of the SARS-CoV-2 S/S2H14 Fab complex structure with three RBDs open shown in two orthogonal orientations. Each SARS-CoV-2 protomer is colored distinctly (cyan, pink, and gold), and N-linked glycans are rendered as dark blue surfaces. The S2H14 light and heavy chain variable domains are colored magenta and purple, respectively. (F) S2H14 binds to an epitope within the SARS-CoV-2 RBM. (G) S2H14 and ACE2 (dark green) bind overlapping RBM epitope. The red star indicates steric clashes. (H) BLI binding competition between S2H14 and ACE2 for binding to the SARS-CoV-2 RBD. (I) Molecular surface representation of the SARS-CoV-2 RBD (gray) with the S2H14 epitope colored by residue conservation across SARS-CoV-2 isolates and SARS-CoV.

Article Snippet: ACE2 Protein, Human , Sino Biological , Cat. No. 10108-H08H.

Techniques: Blocking Assay, Neutralization, Binding Assay

Journal: Cell

Article Title: Mapping Neutralizing and Immunodominant Sites on the SARS-CoV-2 Spike Receptor-Binding Domain by Structure-Guided High-Resolution Serology

doi: 10.1016/j.cell.2020.09.037

Figure Lengend Snippet: The S2A4 mAb Promotes SARS-CoV-2 S Opening through Binding to a Cryptic Epitope (A) SARS-CoV-2 S pseudovirus neutralization assay indicating an IC50 of 3.5 μg/mL. (B and C) Molecular surface representation of the SARS-CoV-2 S/S2A4 Fab complex cryo-EM structure with three RBDs open viewed along two orthogonal orientations. Each SARS-CoV-2 protomer is colored distinctly (cyan, pink, and gold), and N-linked glycans are rendered as dark blue surfaces. The S2A4 light and heavy chains are colored magenta and purple, respectively. (D and E) Zoomed-in views of the contacts formed between S2A4 and the RBD with selected side chains shown. (F) S2A4 and ACE2 (dark green) bind distinct RBD epitopes but would clash via steric hindrance. The red star indicates steric clashes. (G) BLI binding competition between S2A4 and ACE2 for binding to the SARS-CoV-2 RBD. (H) Molecular surface representation of the SARS-CoV-2 RBD (gray) with the S2A4 epitope colored by amino acid residue conservation with SARS-CoV. The position of the SARS-CoV N357 glycan is indicated with red dotted lines.

Article Snippet: ACE2 Protein, Human , Sino Biological , Cat. No. 10108-H08H.

Techniques: Binding Assay, Neutralization, Cryo-EM Sample Prep

Journal: Cell

Article Title: Mapping Neutralizing and Immunodominant Sites on the SARS-CoV-2 Spike Receptor-Binding Domain by Structure-Guided High-Resolution Serology

doi: 10.1016/j.cell.2020.09.037

Figure Lengend Snippet: The S304 mAb Promotes SARS-CoV-2 S Opening through Binding to a Cryptic Epitope Conserved within the Sarbecovirus Subgenus (A and B) Molecular surface representation of the SARS-CoV-2 S/S304 Fab complex cryo-EM structure with three RBDs opened viewed along two orthogonal orientations. Each SARS-CoV-2 S protomer is colored distinctly (cyan, pink, and gold), and N-linked glycans are rendered as dark blue surfaces. The S304 light and heavy chains are colored magenta and purple, respectively. (C) Cryo-EM reconstruction of the S 1 subunit trimer (with disordered S 2 ) bound to three S304 Fabs viewed along two orthogonal orientations and the corresponding atomic model fit in density. Each SARS-CoV-2 S 1 protomer is colored distinctly (cyan, pink, and gold). The S304 light and heavy chains are colored magenta and purple, respectively. (D) Ribbon diagram of the crystal structure of S304 (pink and purple), S2H14, and S309 in complex with the SARS-CoV-2 RBD (light blue). Only the S304 variable domains are shown, whereas S2H14 and S309 were omitted for clarity. (E) Positioning of ACE2 (dark green) relative to the S304 Fab bound to the SARS-CoV-2 RBD. ACE2 N-linked glycans at position N322 and N546 are indicated, as they could putatively clash with S304. (F) Molecular surface representation of the SARS-CoV-2 RBD (gray) with the S304 epitope colored by residue conservation with SARS-CoV. (G and H) Positioning of ACE2 (dark green) relative to the S2A4 (G) and S2X35 (H) Fabs bound to the SARS-CoV-2 RBD. The red stars indicate steric clashes.

Article Snippet: ACE2 Protein, Human , Sino Biological , Cat. No. 10108-H08H.

Techniques: Binding Assay, Cryo-EM Sample Prep

Journal: Cell

Article Title: Mapping Neutralizing and Immunodominant Sites on the SARS-CoV-2 Spike Receptor-Binding Domain by Structure-Guided High-Resolution Serology

doi: 10.1016/j.cell.2020.09.037

Figure Lengend Snippet: Structure-Guided High-Resolution Serology (A) Composite model of the SARS-CoV-2 S trimer with three open RBDs viewed along two orientations with all six mAbs used for competition ELISA shown bound to one RBD. (B–G) Epitopes recognized by each mAb are shown on the surface of the RBD for S2H14 (teal, B), S2H13 (orange, C), S2X35 (red, D), S2A4 (yellow, E), S304 (magenta, F), and S309 (purple, G). The glycan at position N343 is rendered as blue spheres and the RBM is shown as a black outline. (H–J) Competition ELISA (blockade-of-binding) between individual mAbs and sera or plasma from hospitalized (H), symptomatic (I), and asymptomatic (J) COVID-19 convalescent subjects. Each plot shows the magnitude of inhibition of binding to immobilized RBD in the presence of each mAb, expressed as reciprocal sera or plasma dilution blocking 80% of the maximum binding response. (K) Correlation analysis of titers of serum Abs blocking RBD binding to ACE2 and Abs blocking each of the six probe mAbs. (L) Comparison of RBD-specific IgG titers between sera containing Ab blocking at least one probe mAb and sera that do not contain Ab blocking any of the six probe mAbs.

Article Snippet: ACE2 Protein, Human , Sino Biological , Cat. No. 10108-H08H.

Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay, Inhibition, Blocking Assay

Journal: Cell

Article Title: Mapping Neutralizing and Immunodominant Sites on the SARS-CoV-2 Spike Receptor-Binding Domain by Structure-Guided High-Resolution Serology

doi: 10.1016/j.cell.2020.09.037

Figure Lengend Snippet:

Article Snippet: ACE2 Protein, Human , Sino Biological , Cat. No. 10108-H08H.

Techniques: Infection, Recombinant, Blocking Assay, Stable Transfection, Expressing, Magnetic Beads, Protein Binding, Luciferase, Transfection, Plasmid Preparation, Software, Spectrophotometry