Journal: PLoS Biology

Article Title: A Ubiquitin Ligase Complex Regulates Caspase Activation During Sperm Differentiation in Drosophila

doi: 10.1371/journal.pbio.0050251

Figure Lengend Snippet: (A–H) Visualization of active drICE with anti-cleaved caspase-3 antibody (CM1; green) and axonemal tubulin polyglycylation with anti-glycylated tubulin monoclonal antibody (AXO 49; red). These figures are composed of a green layer only in the left panel, and green and red layers combined in the right panel. (A) Wild-type individualizing spermatids stain positively for active effector caspase and polyglycylated axonemal tubulin (white arrows pointing at cystic bulges [CBs] and red arrow pointing at a waste bag [WB]). Elongated spermatids from (B) homozygotes for the null cul3 mds1 allele or (C and D) transheterozygotes for cul3 mds1 and two different deficiencies that cover the cullin-3 gene, DF (2L)ED3 and DF (2L)Exel8034, respectively, stain for polyglycylation but not for active effector caspase. (E–G) Homozygote mutants for three hypomorphic cul3 Testis alleles, cul3 mds5 , cul3 mds3 , and cul3 mds4 , respectively, have spermatid individualization defects but still display some levels of active effector caspase expression. (H) However, the level of active effector caspase expression was dramatically reduced in spermatids from transheterozygote mutants for the null cul3 mds1 and either of the hypomorphic alleles, such as cul3 mds4 . All the figures are in the same magnification; scale bar 200 μm. (I) The diagram depicts a DEVDase activity assay for cul3 mds1 −/− testes. Caspase-3–like (DEVDase) activity is detected in wild-type testes and is blocked either after treatment with the caspase-3 inhibitor Z-VAD.fmk or in cul3 mds1 −/− testes. DEVDase activity, presented as relative luminescence units (RLUs), was determined on Ac-DEVD-pNA substrate in testis extracts made of 180 wild-type ( yw ) or cul3 mds1 −/− testes treated with Z-VAD or left untreated (DMSO). Readings were obtained every 2 min, and each time interval represents an average (mean ± SEM) of five readings. Note that the level of DEVDase activity in cul3 mds1 −/− testes is highly similar to the corresponding level in wild-type testes that were treated with Z-VAD. (J) A Western blot analysis for the assessment of the relative protein amounts used in (I). A portion of the testis extracts in (I) were used as controls to determine the relative amounts of total protein in each extract using the anti- β -Tubulin antibody.

Article Snippet: The tubes with 10 μl of the testes extracts were used for Western blot analysis to control for the protein amount in the samples by probing with anti- β -tubulin antibody (E7; 1:1000; Hybridoma Bank; http://dshb.biology.uiowa.edu/ ).

Techniques: Staining, Expressing, Activity Assay, Western Blot

Journal: PLoS Biology

Article Title: A Ubiquitin Ligase Complex Regulates Caspase Activation During Sperm Differentiation in Drosophila

doi: 10.1371/journal.pbio.0050251

Figure Lengend Snippet: (A) Diap1 protein levels were not affected in cul3 mds1 and klhl10 3 mutant testes, as assessed by Western blotting of protein extracts from dissected testes. Therefore, Diap1 does not appear to be a major target for the Cul3-based E3-ligase complex. β - tubulin protein levels served as loading control. (B andC) Co-IP experiment in S2 cells indicate that Klhl10 can bind to the BIR domain of dBruce. The immunoprecipitate (IP) is shown at the top, and pre-incubation of whole lysates are shown at the bottom (Input). Cell lysates were incubated with IgG beads which bind to Protein A (PrA). For Western blotting of IPs, (B) anti-dBruce antibody or (C) anti-HA antibody were used. (B) Cells were co-transfected with a dbruce “mini gene” (consisting of the first N-terminal 1,622 amino acids, including the BIR domain, and the last C-terminal 446 amino acids that contain the UBC domain) and (lane 1) PrA-klhl10 or (lane 2) PrA-GFP (see for details). (C) Cells were co-transfected with HA-tagged dBruce-BIR peptide containing the first N-terminal 387 amino acids of dBruce that includes the BIR domain region (amino acids 251–321). This motif is sufficient to bind to Klhl10 in S2 cells (see for details).

Article Snippet: The tubes with 10 μl of the testes extracts were used for Western blot analysis to control for the protein amount in the samples by probing with anti- β -tubulin antibody (E7; 1:1000; Hybridoma Bank; http://dshb.biology.uiowa.edu/ ).

Techniques: Mutagenesis, Western Blot, Control, Co-Immunoprecipitation Assay, Incubation, Transfection

Journal: Precision medicine

Article Title: The mirror RNA expression pattern in human tissues

doi: 10.14800/pm.1036

Figure Lengend Snippet: The percentages of mirror RNAs in eight tissues are shown at Chr7q11.23. (a) mirror, (b) unidirectional expression. The expression activities in various tissues are grouped by the expression levels.

Article Snippet: Samples and RNA Preparation Human RNAs from artery, vein, bone marrow, lymph node, small intestine, adrenal glands, pancreas, prostate, thymus, adipose, brain cortex, heart, skin, skeletal muscle, prostate (50 g) were purchased from Clontech and US Biological [ 22 , 23 ] .

Techniques: Expressing

Journal: Precision medicine

Article Title: The mirror RNA expression pattern in human tissues

doi: 10.14800/pm.1036

Figure Lengend Snippet: The results at chromosomal loci 7q11.23 are shown. For example, 72% of mirror RNAs was expressed in all of the eight tissues under the study, and only 10% of these mirror RNAs was observed in only one tissue.

Article Snippet: Samples and RNA Preparation Human RNAs from artery, vein, bone marrow, lymph node, small intestine, adrenal glands, pancreas, prostate, thymus, adipose, brain cortex, heart, skin, skeletal muscle, prostate (50 g) were purchased from Clontech and US Biological [ 22 , 23 ] .

Techniques:

Journal: Precision medicine

Article Title: The mirror RNA expression pattern in human tissues

doi: 10.14800/pm.1036

Figure Lengend Snippet: Four known protein-coding genes are shown. (a) PDE4D, (b) ELOVL7, (c) DEPDC1B, and (d) GTF2IRD2. Each bar represents a percentage of mirror (blue) or unidirection (red) in total RNAs.

Article Snippet: Samples and RNA Preparation Human RNAs from artery, vein, bone marrow, lymph node, small intestine, adrenal glands, pancreas, prostate, thymus, adipose, brain cortex, heart, skin, skeletal muscle, prostate (50 g) were purchased from Clontech and US Biological [ 22 , 23 ] .

Techniques:

Journal: Precision medicine

Article Title: The mirror RNA expression pattern in human tissues

doi: 10.14800/pm.1036

Figure Lengend Snippet: (a) introns; (b) intergenic regions. Each bar represents a percentage of mirror (blue) or unidirection (red) in total RNAs.

Article Snippet: Samples and RNA Preparation Human RNAs from artery, vein, bone marrow, lymph node, small intestine, adrenal glands, pancreas, prostate, thymus, adipose, brain cortex, heart, skin, skeletal muscle, prostate (50 g) were purchased from Clontech and US Biological [ 22 , 23 ] .

Techniques:

Journal: Precision medicine

Article Title: The mirror RNA expression pattern in human tissues

doi: 10.14800/pm.1036

Figure Lengend Snippet: Each bar represents a percentage of mirror (blue) or unidirection (red) in total RNAs in the 5′-flanking regions of 1-kb, 2-kb and 10-kb upstream to the transcription start site (TSS).

Article Snippet: Samples and RNA Preparation Human RNAs from artery, vein, bone marrow, lymph node, small intestine, adrenal glands, pancreas, prostate, thymus, adipose, brain cortex, heart, skin, skeletal muscle, prostate (50 g) were purchased from Clontech and US Biological [ 22 , 23 ] .

Techniques:

Journal: Precision medicine

Article Title: The mirror RNA expression pattern in human tissues

doi: 10.14800/pm.1036

Figure Lengend Snippet: Each bar represents a percentage of mirror (blue) or unidirection (red) in total RNAs in the repetitive elements in human genome. The types of repeats are labeled above each figure.

Article Snippet: Samples and RNA Preparation Human RNAs from artery, vein, bone marrow, lymph node, small intestine, adrenal glands, pancreas, prostate, thymus, adipose, brain cortex, heart, skin, skeletal muscle, prostate (50 g) were purchased from Clontech and US Biological [ 22 , 23 ] .

Techniques: Labeling

Journal: Cell reports

Article Title: Platelet-instructed SPP1 + macrophages drive myofibroblast activation in fibrosis in a CXCL4-dependent manner.

doi: 10.1016/j.celrep.2023.112131

Figure Lengend Snippet: Figure 7. SPP1+ profibrotic macrophages expand in human CKD and heart failure (A) UMAP embedding of 4,404 mononuclear phagocytes sub-clustered from CD10 single cells from 15 human kidneys by Kuppe et al.6 Labels refer to clusters. cDC, conventional dendritic cells; Mono, monocytes; Res-like Mac, resident-like macrophages; SPP1 Mac, SPP1+ macrophages. (B) Bar plot of cluster cell numbers in CKD versus healthy kidneys after normalization via Log2 transformation. Log2FC, log 2-Fold Change. (C) RNA-ISH for SPP1 and COL1A1 combined with immunofluorescent CD68 staining in human kidney nephrectomies. SPP1+CD68+ macrophages are circled in white. Scale bar = 30 mm. (D) Pearson correlation of the number of COL1A1+ fibroblasts with SPP1+CD68+ macrophages in human kidney nephrectomies (n = 41). (E) UMAP embedding of 20,892 mononuclear phagocytes sub-clustered from CD45+ single cells from six human heart samples from Rao et al.50 Labels refer to clusters. Inflam. Mac, inflammatory macrophages. (F) Bar plot of cluster cell numbers in heart failure versus healthy hearts after normalization via Log2 transformation. Log2FC, log 2-Fold Change. (G) Cardiac ECM regulator score stratified by immune cell type. For (B) and (F), Fisher’s exact test was computed using false discovery rate correction for multiple testing. For (G), a two-tailed unpaired t test was performed. ***p < 0.001, ****p < 0.0001. See also Figure S7.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Anti-mouse CD11b (APC) eBioscience Cat# 17-0112-83; RRID: AB_469344 Anti-mouse mCD25 (PE-Cy7) Biolegend Cat# 102016; RRID: AB_312865 Anti-mouse CD4 (PB) Biolegend Cat# 100428; RRID: AB_493647 Anti-human CD68 Agilent Cat# M0876; RRID: AB_2074844 Anti-mouse-Fc (AF488) Jackson ImmunoResearch Cat# 715-545-151; RRID: AB_2341099 mUromoduline abcam Cat# ab207170; RRID: AB_2889163 mPDGFRa R&D Systems Cat# AF1062; RRID: AB_2236897 LTL-Fitc Vector Labs Cat# FL1321; RRID: AB_2336559 mKIM1 R&D Systems Cat#: AF1817; RRID: AB_2116446 mCD68 abcam Cat# ab53444; RRID: AB_869007 Anti-rabbit-Fc (AF647) Dianova Cat# 111-605-008; RRID: AB_2338074 Anti-goat-Fc (Cy3) Dianova Cat# 705-165-147; RRID: AB_2307351 Anti-goat-Fc (AF647) Dianova Cat# 705-605-147; RRID: AB_2340437 Anti-rat-Fc (AF647) Dianova Cat# 712-605-153; RRID: AB_2340694 Bacterial and virus strains pBABE-puro SV40 LT vector Addgene #13970 Biological samples Human nephrectomy tissue samples (healthy and CKD) This paper N/A Chemicals, peptides, and recombinant proteins LPS Sigma-Aldrich L4391-1MG Thrombine Molecular-Innovations MTHROM-0.05MG Critical commercial assays RNA-ScopeTM Multiplex Fluorescent V2 Assay ACD 323100 10x genomics single nuclear RNA-seq kit 10x genomics 1000077 Pikro Siriusred staining kit Morphisto 13422 Cell Tracker CMFDA Dye Thermo Fisher C2925 Cell Tracker CMTPX Dye Thermo Fisher C34552 CD11b-Microbeads Miltenyi Biotech 130-049-601 CD117-Microbeads Miltenyi Biotech 130-091-224 High-Capacity cDNA Reverse Transcription Kit Thermo Fisher 43-688-13 iTaq Univer SYBR Green Supermix Biorad 1725125 Deposited data Data scRNAseq MI hearts (murine) Forte et al.23 E-MTAB-7895 Data of human healthy and CKD kidney Kuppe et al.6 10.5281/zenodo.4059315 Data of human heart samples Rao et al.50 GSE145154 Reference mapping onto Dataset of Sanin et al. Sanin et al.38 GSE171328, GSE157313 Reference mapping of snRNASeq Data of IRI kidney onto murine IRI-snRNASeq dataset Kirita et al.41 GSE139107 (Continued on next page) Cell Reports 42, 112131, February 28, 2023 17

Techniques: Transformation Assay, Staining, Two Tailed Test

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