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  • 94
    Alomone Labs anti hvcn1 antibody
    Anti Hvcn1 Antibody, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti hvcn1 antibody/product/Alomone Labs
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti hvcn1 antibody - by Bioz Stars, 2022-09
    94/100 stars
      Buy from Supplier

    86
    Cell Signaling Technology Inc anti hvcn1 antibody
    Human <t>HVCN1</t> mediates CC30 and CC45 LukAB binding and cytotoxicity. A: Intoxication of CHO cells expressing firefly luciferase ( Fluc ) or HVCN1 with CC30 and CC45 LukAB. Cell viability was measured with Cell Titer. Data from three independent experiments are represented as mean values ±SD. For each toxin, statistical significance was determined by two-way ANOVA (****, P ≤ 0.0001; ns, not significant). B: Binding of biotinylated CC30 and CC45 LukAB to CHO cells expressing Fluc or HVCN1 . Binding was measured by PerCP/Cy5.5 streptavidin staining. Data from three independent experiments are represented as mean values ±SD. For each toxin, statistical significance was determined by two-way ANOVA (****, P ≤ 0.0001; ***, P ≤ 0.001; ns, not significant). C: Binding of biotinylated CC30 and CC45 LukAB (3 μg/ml) to CHO cells transduced with Fluc or HVCN1 in the presence of the indicated excess of unlabeled toxins. Binding was measured by PerCP/Cy5.5 streptavidin staining. Data from three independent experiments are represented as mean values ±SD. D: Pull-down of purified His-tagged LukAB or LukSF with Strep-tagged HVCN1. Input represents resin-bound ligand (HVCN1 or TBS control) and toxin binding partner (LukAB or LukSF). Flow-through (FT), wash, and elution lanes represent fractions from the pull-down after toxin binding (see Methods ). Top panel is Sypro Ruby stained SDS-PAGE, middle panel is an immunoblot to detect the toxins, and bottom panel is an immunoblot to detect HVCN1. Representative images of two independent experiments are shown. E: Intoxication of primary human B cells, CD4-T and CD8-T cells with indicated concentrations of CC30 and CC45 LukAB. Membrane damage was detected using Fixable Viability Dye eFluor ™ 450. Data from cells isolated from four different donors are represented as mean values ±SEM. Also refer to Extended Data Figure 4 .
    Anti Hvcn1 Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti hvcn1 antibody/product/Cell Signaling Technology Inc
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti hvcn1 antibody - by Bioz Stars, 2022-09
    86/100 stars
      Buy from Supplier

    Image Search Results


    Human HVCN1 mediates CC30 and CC45 LukAB binding and cytotoxicity. A: Intoxication of CHO cells expressing firefly luciferase ( Fluc ) or HVCN1 with CC30 and CC45 LukAB. Cell viability was measured with Cell Titer. Data from three independent experiments are represented as mean values ±SD. For each toxin, statistical significance was determined by two-way ANOVA (****, P ≤ 0.0001; ns, not significant). B: Binding of biotinylated CC30 and CC45 LukAB to CHO cells expressing Fluc or HVCN1 . Binding was measured by PerCP/Cy5.5 streptavidin staining. Data from three independent experiments are represented as mean values ±SD. For each toxin, statistical significance was determined by two-way ANOVA (****, P ≤ 0.0001; ***, P ≤ 0.001; ns, not significant). C: Binding of biotinylated CC30 and CC45 LukAB (3 μg/ml) to CHO cells transduced with Fluc or HVCN1 in the presence of the indicated excess of unlabeled toxins. Binding was measured by PerCP/Cy5.5 streptavidin staining. Data from three independent experiments are represented as mean values ±SD. D: Pull-down of purified His-tagged LukAB or LukSF with Strep-tagged HVCN1. Input represents resin-bound ligand (HVCN1 or TBS control) and toxin binding partner (LukAB or LukSF). Flow-through (FT), wash, and elution lanes represent fractions from the pull-down after toxin binding (see Methods ). Top panel is Sypro Ruby stained SDS-PAGE, middle panel is an immunoblot to detect the toxins, and bottom panel is an immunoblot to detect HVCN1. Representative images of two independent experiments are shown. E: Intoxication of primary human B cells, CD4-T and CD8-T cells with indicated concentrations of CC30 and CC45 LukAB. Membrane damage was detected using Fixable Viability Dye eFluor ™ 450. Data from cells isolated from four different donors are represented as mean values ±SEM. Also refer to Extended Data Figure 4 .

    Journal: Nature microbiology

    Article Title: Genetic variation of staphylococcal LukAB toxin determines receptor tropism

    doi: 10.1038/s41564-021-00890-3

    Figure Lengend Snippet: Human HVCN1 mediates CC30 and CC45 LukAB binding and cytotoxicity. A: Intoxication of CHO cells expressing firefly luciferase ( Fluc ) or HVCN1 with CC30 and CC45 LukAB. Cell viability was measured with Cell Titer. Data from three independent experiments are represented as mean values ±SD. For each toxin, statistical significance was determined by two-way ANOVA (****, P ≤ 0.0001; ns, not significant). B: Binding of biotinylated CC30 and CC45 LukAB to CHO cells expressing Fluc or HVCN1 . Binding was measured by PerCP/Cy5.5 streptavidin staining. Data from three independent experiments are represented as mean values ±SD. For each toxin, statistical significance was determined by two-way ANOVA (****, P ≤ 0.0001; ***, P ≤ 0.001; ns, not significant). C: Binding of biotinylated CC30 and CC45 LukAB (3 μg/ml) to CHO cells transduced with Fluc or HVCN1 in the presence of the indicated excess of unlabeled toxins. Binding was measured by PerCP/Cy5.5 streptavidin staining. Data from three independent experiments are represented as mean values ±SD. D: Pull-down of purified His-tagged LukAB or LukSF with Strep-tagged HVCN1. Input represents resin-bound ligand (HVCN1 or TBS control) and toxin binding partner (LukAB or LukSF). Flow-through (FT), wash, and elution lanes represent fractions from the pull-down after toxin binding (see Methods ). Top panel is Sypro Ruby stained SDS-PAGE, middle panel is an immunoblot to detect the toxins, and bottom panel is an immunoblot to detect HVCN1. Representative images of two independent experiments are shown. E: Intoxication of primary human B cells, CD4-T and CD8-T cells with indicated concentrations of CC30 and CC45 LukAB. Membrane damage was detected using Fixable Viability Dye eFluor ™ 450. Data from cells isolated from four different donors are represented as mean values ±SEM. Also refer to Extended Data Figure 4 .

    Article Snippet: After blocking in 5% milk in PBS-T for 1 h at room temperature, membranes were incubated with primary antibodies ((anti-HVCN1 (1:200, AHC-001, Alomone Labs), and anti-β-Actin (1:1000, 715 8H10D10, Cell Signaling Technology)) diluted in 5% milk in PBS-T for 16 h at 4°C.

    Techniques: Binding Assay, Expressing, Luciferase, Staining, Transduction, Purification, SDS Page, Isolation

    CC30 S. aureus kills leukocytes in a LukAB and HVCN1 dependent manner. A-B: PCR targeting lukA and hlgA (A) and immunoblot of CC30 LukAB in supernatants of wild type and Δ lukAB CC30 S. aureus 62300D1 (B). Asterisks indicate non-specific bands that serve as loading controls. One replicate of this experiment was performed (A). Representative image of two independent experiments is shown (B). C: Viability of human PMNs following a 2-h infection with nonopsonized wild type (WT) or isogenic Δ lukAB CC30 S. aureus 62300D1 at the indicated multiplicity of infection (MOI). PMN lysis measured by LDH release. Data are from PMNs isolated from six independent donors represented as the mean values ±SEM. Statistical significance was determined by two-way ANOVA (****, P ≤ 0.0001; #, P = 0.0119). D-E: Viability of ITGAM shRNA THP1 cells transduced with lentiCRISPRv2 expressing non-targeting (nt) sgRNA or HVCN1 sgRNA and infected with nonopsonized (extracellular infection, D) or with opsonized (intracellular conditions, E) WT and Δ lukAB CC30 S. aureus 62300D1 for 2h (MOI=100). THP1 cell lysis was measured by LDH release. Data from three independent experiments are represented as the mean ±SD. Statistical significance was determined by t-test (two-tailed), numbers indicate P values.

    Journal: Nature microbiology

    Article Title: Genetic variation of staphylococcal LukAB toxin determines receptor tropism

    doi: 10.1038/s41564-021-00890-3

    Figure Lengend Snippet: CC30 S. aureus kills leukocytes in a LukAB and HVCN1 dependent manner. A-B: PCR targeting lukA and hlgA (A) and immunoblot of CC30 LukAB in supernatants of wild type and Δ lukAB CC30 S. aureus 62300D1 (B). Asterisks indicate non-specific bands that serve as loading controls. One replicate of this experiment was performed (A). Representative image of two independent experiments is shown (B). C: Viability of human PMNs following a 2-h infection with nonopsonized wild type (WT) or isogenic Δ lukAB CC30 S. aureus 62300D1 at the indicated multiplicity of infection (MOI). PMN lysis measured by LDH release. Data are from PMNs isolated from six independent donors represented as the mean values ±SEM. Statistical significance was determined by two-way ANOVA (****, P ≤ 0.0001; #, P = 0.0119). D-E: Viability of ITGAM shRNA THP1 cells transduced with lentiCRISPRv2 expressing non-targeting (nt) sgRNA or HVCN1 sgRNA and infected with nonopsonized (extracellular infection, D) or with opsonized (intracellular conditions, E) WT and Δ lukAB CC30 S. aureus 62300D1 for 2h (MOI=100). THP1 cell lysis was measured by LDH release. Data from three independent experiments are represented as the mean ±SD. Statistical significance was determined by t-test (two-tailed), numbers indicate P values.

    Article Snippet: After blocking in 5% milk in PBS-T for 1 h at room temperature, membranes were incubated with primary antibodies ((anti-HVCN1 (1:200, AHC-001, Alomone Labs), and anti-β-Actin (1:1000, 715 8H10D10, Cell Signaling Technology)) diluted in 5% milk in PBS-T for 16 h at 4°C.

    Techniques: Polymerase Chain Reaction, Infection, Lysis, Isolation, shRNA, Transduction, Expressing, Two Tailed Test

    Flow cytometry gating (part 1) A: Flow cytometry gating scheme utilized to measure surface CD11b levels in scramble shRNA ( top ) and ITGAM shRNA ( bottom ) expressing THP1 cell ( Figure 2A ) using APC-conjugated anti-CD11b antibody. B: Flow cytometry gating scheme utilized to measure binding of biotinylated LukAB (CC30 LukAB is shown as an example) to CHO cells expressing Fluc ( top ) or HVCN1 ( bottom ) using PerCP/Cy5.5-conjugated streptavidin staining ( Figures 5B – 5C ). C: Flow cytometry gating scheme utilized to measure membrane damage in B cells following treatment with PBS control ( top ) and LukAB (CC30 LukAB is shown as an example, bottom ) using Fixable Viability Dye eFluor ™ 450 ( Figure 5E ). D-E: Flow cytometry gating scheme utilized to measure membrane damage in CD4-positive (D) and CD8-positive (E) T cells following treatment with PBS control ( top ) and LukAB (CC30 LukAB is shown as an example, bottom ) using Fixable Viability Dye eFluor ™ 450 ( Figure 5E ).

    Journal: Nature microbiology

    Article Title: Genetic variation of staphylococcal LukAB toxin determines receptor tropism

    doi: 10.1038/s41564-021-00890-3

    Figure Lengend Snippet: Flow cytometry gating (part 1) A: Flow cytometry gating scheme utilized to measure surface CD11b levels in scramble shRNA ( top ) and ITGAM shRNA ( bottom ) expressing THP1 cell ( Figure 2A ) using APC-conjugated anti-CD11b antibody. B: Flow cytometry gating scheme utilized to measure binding of biotinylated LukAB (CC30 LukAB is shown as an example) to CHO cells expressing Fluc ( top ) or HVCN1 ( bottom ) using PerCP/Cy5.5-conjugated streptavidin staining ( Figures 5B – 5C ). C: Flow cytometry gating scheme utilized to measure membrane damage in B cells following treatment with PBS control ( top ) and LukAB (CC30 LukAB is shown as an example, bottom ) using Fixable Viability Dye eFluor ™ 450 ( Figure 5E ). D-E: Flow cytometry gating scheme utilized to measure membrane damage in CD4-positive (D) and CD8-positive (E) T cells following treatment with PBS control ( top ) and LukAB (CC30 LukAB is shown as an example, bottom ) using Fixable Viability Dye eFluor ™ 450 ( Figure 5E ).

    Article Snippet: After blocking in 5% milk in PBS-T for 1 h at room temperature, membranes were incubated with primary antibodies ((anti-HVCN1 (1:200, AHC-001, Alomone Labs), and anti-β-Actin (1:1000, 715 8H10D10, Cell Signaling Technology)) diluted in 5% milk in PBS-T for 16 h at 4°C.

    Techniques: Flow Cytometry, shRNA, Expressing, Binding Assay, Staining

    Related to Figure 5 . Consensus human blood cell type expression of HVCN1 derived from RNA-seq data from internally generated Human Protein Atlas (HPA) data 1 . Transcript expression values are presented as Normalized eXpression (NX), resulting from the internal normalization pipeline for 18 blood cell types and total peripheral blood mononuclear cells (PBMC). Data is available at v20.proteinatlas.org/ENSG00000122986-HVCN1/blood , Human Protein Atlas available from www.proteinatlas.org 34

    Journal: Nature microbiology

    Article Title: Genetic variation of staphylococcal LukAB toxin determines receptor tropism

    doi: 10.1038/s41564-021-00890-3

    Figure Lengend Snippet: Related to Figure 5 . Consensus human blood cell type expression of HVCN1 derived from RNA-seq data from internally generated Human Protein Atlas (HPA) data 1 . Transcript expression values are presented as Normalized eXpression (NX), resulting from the internal normalization pipeline for 18 blood cell types and total peripheral blood mononuclear cells (PBMC). Data is available at v20.proteinatlas.org/ENSG00000122986-HVCN1/blood , Human Protein Atlas available from www.proteinatlas.org 34

    Article Snippet: After blocking in 5% milk in PBS-T for 1 h at room temperature, membranes were incubated with primary antibodies ((anti-HVCN1 (1:200, AHC-001, Alomone Labs), and anti-β-Actin (1:1000, 715 8H10D10, Cell Signaling Technology)) diluted in 5% milk in PBS-T for 16 h at 4°C.

    Techniques: Expressing, Derivative Assay, RNA Sequencing Assay, Generated

    Identification of HVCN1 as a cellular target for the CC30 and CC45 LukAB variants. A: Schematic of the CC30 LukAB GeCKO screen in ITGAM shRNA THP1 cells. B: Enrichment of specific sgRNAs from the GeCKO library following two rounds of CC30 LukAB selection. Data are presented as the number of sgRNAs significantly enriched in the intoxicated sample versus the average fold enrichment as compared to untreated control. C: Intoxication of ITGAM shRNA THP1 cells transduced with lentiCRISPRv2 expressing sgRNAs targeting indicated genes with CC30 LukAB. Cell viability was measured with Cell Titer. Data are represented as the average of two independent experiments each performed in duplicate. D: Gel image of T7 Endonuclease I-treated HVCN1 PCR products confirming HVCN1 targeting by the sgRNA. HVCN1 was amplified from genomic DNA of ITGAM shRNA THP1 cells transduced with lentiCRISPRv2 expressing non-targeting (nt) sgRNA or HVCN1 sgRNA. Asterisks indicate T7 Endonuclease I cleavage bands. One replicate of this experiment was performed. E: Immunoblot of HVCN1 in ITGAM shRNA THP1 cells transduced with lentiCRISPRv2 expressing non-targeting (nt) sgRNA or HVCN1 sgRNA. Anti-actin immunoblot is shown below as a loading control. Representative image of three independent experiments is shown. F: Intoxication of ITGAM shRNA THP1 cells transduced with lentiCRISPRv2 expressing non-targeting (nt) sgRNA or HVCN1 sgRNA with indicated concentration of CC30 LukAB, CC45 LukAB, and HlgAB. Cell viability was measured with Cell Titer. Data from three independent experiments are represented as mean values ±SD. For each toxin, statistical significance was determined by two-way ANOVA (****, P ≤ 0.0001; $, P = 0.0021; #, P = 0.0072; ns, not significant, > 0.9999). Also refer to Supplementary Table 3 .

    Journal: Nature microbiology

    Article Title: Genetic variation of staphylococcal LukAB toxin determines receptor tropism

    doi: 10.1038/s41564-021-00890-3

    Figure Lengend Snippet: Identification of HVCN1 as a cellular target for the CC30 and CC45 LukAB variants. A: Schematic of the CC30 LukAB GeCKO screen in ITGAM shRNA THP1 cells. B: Enrichment of specific sgRNAs from the GeCKO library following two rounds of CC30 LukAB selection. Data are presented as the number of sgRNAs significantly enriched in the intoxicated sample versus the average fold enrichment as compared to untreated control. C: Intoxication of ITGAM shRNA THP1 cells transduced with lentiCRISPRv2 expressing sgRNAs targeting indicated genes with CC30 LukAB. Cell viability was measured with Cell Titer. Data are represented as the average of two independent experiments each performed in duplicate. D: Gel image of T7 Endonuclease I-treated HVCN1 PCR products confirming HVCN1 targeting by the sgRNA. HVCN1 was amplified from genomic DNA of ITGAM shRNA THP1 cells transduced with lentiCRISPRv2 expressing non-targeting (nt) sgRNA or HVCN1 sgRNA. Asterisks indicate T7 Endonuclease I cleavage bands. One replicate of this experiment was performed. E: Immunoblot of HVCN1 in ITGAM shRNA THP1 cells transduced with lentiCRISPRv2 expressing non-targeting (nt) sgRNA or HVCN1 sgRNA. Anti-actin immunoblot is shown below as a loading control. Representative image of three independent experiments is shown. F: Intoxication of ITGAM shRNA THP1 cells transduced with lentiCRISPRv2 expressing non-targeting (nt) sgRNA or HVCN1 sgRNA with indicated concentration of CC30 LukAB, CC45 LukAB, and HlgAB. Cell viability was measured with Cell Titer. Data from three independent experiments are represented as mean values ±SD. For each toxin, statistical significance was determined by two-way ANOVA (****, P ≤ 0.0001; $, P = 0.0021; #, P = 0.0072; ns, not significant, > 0.9999). Also refer to Supplementary Table 3 .

    Article Snippet: After blocking in 5% milk in PBS-T for 1 h at room temperature, membranes were incubated with primary antibodies ((anti-HVCN1 (1:200, AHC-001, Alomone Labs), and anti-β-Actin (1:1000, 715 8H10D10, Cell Signaling Technology)) diluted in 5% milk in PBS-T for 16 h at 4°C.

    Techniques: shRNA, Selection, Transduction, Expressing, Polymerase Chain Reaction, Amplification, Concentration Assay

    Related to Figure 6 . A: Schematic representation of murine Hvcn1 locus and DNA template used to humanize exon 4. B: Genotyping strategy using genomic DNA isolated from wild type (WT), heterozygous (het), and homozygous (homo) hHVCN1 mice using primers VJT2065 and VJT2069. Images are representative of multiple independent experiments as routinely performed for hHVCN1 mouse genotyping. C-G: CFUs in the kidneys (C), livers (D), hearts (E), spleens (F), and lungs (G) collected from WT and hHVCN1 mice infected intravenously with 1×10 7 CFU of lukAB -deficient USA300 strain LAC. Data from 11 WT and 10 hHVCN1 mice are represented as mean values ±SEM. Statistical significance was determined by t-test (two-tailed), numbers above bars indicate P values. H-K: CFUs in the livers (H), hearts (I), spleens (J), and lungs (K) collected from WT and hHVCN1 mice infected intravenously with 5–10×10 7 CFU CC30 S. aureus MUZ211 (CFU obtained from 11 WT and 24 hHVCN1 mice) and 62300D1 (CFU obtained from 11 WT and 10 hHVCN1 mice). Data for each isolate are from mice infected over three independent experiments and is represented as mean values ±SEM. Statistical significance was determined by t-test (two-tailed), numbers above bars indicate P values.

    Journal: Nature microbiology

    Article Title: Genetic variation of staphylococcal LukAB toxin determines receptor tropism

    doi: 10.1038/s41564-021-00890-3

    Figure Lengend Snippet: Related to Figure 6 . A: Schematic representation of murine Hvcn1 locus and DNA template used to humanize exon 4. B: Genotyping strategy using genomic DNA isolated from wild type (WT), heterozygous (het), and homozygous (homo) hHVCN1 mice using primers VJT2065 and VJT2069. Images are representative of multiple independent experiments as routinely performed for hHVCN1 mouse genotyping. C-G: CFUs in the kidneys (C), livers (D), hearts (E), spleens (F), and lungs (G) collected from WT and hHVCN1 mice infected intravenously with 1×10 7 CFU of lukAB -deficient USA300 strain LAC. Data from 11 WT and 10 hHVCN1 mice are represented as mean values ±SEM. Statistical significance was determined by t-test (two-tailed), numbers above bars indicate P values. H-K: CFUs in the livers (H), hearts (I), spleens (J), and lungs (K) collected from WT and hHVCN1 mice infected intravenously with 5–10×10 7 CFU CC30 S. aureus MUZ211 (CFU obtained from 11 WT and 24 hHVCN1 mice) and 62300D1 (CFU obtained from 11 WT and 10 hHVCN1 mice). Data for each isolate are from mice infected over three independent experiments and is represented as mean values ±SEM. Statistical significance was determined by t-test (two-tailed), numbers above bars indicate P values.

    Article Snippet: After blocking in 5% milk in PBS-T for 1 h at room temperature, membranes were incubated with primary antibodies ((anti-HVCN1 (1:200, AHC-001, Alomone Labs), and anti-β-Actin (1:1000, 715 8H10D10, Cell Signaling Technology)) diluted in 5% milk in PBS-T for 16 h at 4°C.

    Techniques: Genotyping Assay, Isolation, Mouse Assay, Infection, Two Tailed Test

    Flow cytometry gating (part 2) A: Flow cytometry gating scheme utilized to measure membrane damage in PECs after treatment with PBS control ( top ) and leukocidins (LukED is shown as an example, bottom ) using Fixable Viability Dye eFluor ™ 450 ( Figure 6A ). B: Flow cytometry gating scheme utilized to measure membrane damage in Lenti-X 293T cells expressing C-terminal GFP-tagged wildtype HVCN1 and chimeric proteins (human HVCN1 is shown as an example) following treatment with PBS control ( top ) and CC30 LukAB ( bottom ) using Fixable Viability Dye eFluor ™ 450 ( Figure 6D ).

    Journal: Nature microbiology

    Article Title: Genetic variation of staphylococcal LukAB toxin determines receptor tropism

    doi: 10.1038/s41564-021-00890-3

    Figure Lengend Snippet: Flow cytometry gating (part 2) A: Flow cytometry gating scheme utilized to measure membrane damage in PECs after treatment with PBS control ( top ) and leukocidins (LukED is shown as an example, bottom ) using Fixable Viability Dye eFluor ™ 450 ( Figure 6A ). B: Flow cytometry gating scheme utilized to measure membrane damage in Lenti-X 293T cells expressing C-terminal GFP-tagged wildtype HVCN1 and chimeric proteins (human HVCN1 is shown as an example) following treatment with PBS control ( top ) and CC30 LukAB ( bottom ) using Fixable Viability Dye eFluor ™ 450 ( Figure 6D ).

    Article Snippet: After blocking in 5% milk in PBS-T for 1 h at room temperature, membranes were incubated with primary antibodies ((anti-HVCN1 (1:200, AHC-001, Alomone Labs), and anti-β-Actin (1:1000, 715 8H10D10, Cell Signaling Technology)) diluted in 5% milk in PBS-T for 16 h at 4°C.

    Techniques: Flow Cytometry, Expressing

    LukAB targeting of HVCN1 promotes S. aureus pathogenesis. A: Intoxication of murine PECs with indicated concentrations of leukocidins. Membrane damage was detected using Fixable Viability Dye eFluor ™ 450. Data are represented as the average of three independent experiments ± SEM B: ( inset ) Immunoblot of HVCN1 in CHO cells expressing firefly luciferase (Fluc), human (HVCN1) or murine (mHVCN1) HVCN1. Anti-actin immunoblot is shown above as a loading control. Representative images of four independent samples from one immunoblot are shown, see corresponding Source Data for full gel. Numbers on the left indicate migration of the corresponding molecular weight standards (in kDa). Target protein levels normalized by actin were obtained using ImageJ from four independent protein samples: HVCN1 = 0.310 ± 0.111, mHVCN1 = 0.333 ± 0.066 (mean ± SD), P = 0.742 as determined by unpaired t test. ( main figure ) Intoxication of Fluc, HVCN1, and mHVCN1 expressing CHO cells with indicated concentrations of CC30 LukAB. Cell viability was measured with Cell Titer. Data from three independent experiments are represented as mean values ±SD. Statistical significance was determined by two-way ANOVA (****, P ≤ 0.0001; ns, not significant). C: Schematic architecture of HVCN1 and the amino acid alignments of human and murine extracellular loops generated using Clustal Omega. D: Intoxication of Lenti-X 293T cells expressing C-terminal GFP-tagged human, murine, and chimeric HVCN1 proteins with indicated concentrations of CC30 LukAB. Membrane damage was detected using Fixable Viability Dye eFluor ™ 450. Data from three independent experiments are represented as the mean values ±SD. Statistical significance was determined by two-way ANOVA (****, P ≤ 0.0001; **, P ≤ 0.01; ns, not significant). E: Intoxication of PECs from wild type (WT) and hHVCN1 mice with indicated LukAB. Membrane damage was detected using Propidium Iodide (PI) incorporation. Data from five mice per genotype over three independent experiments are represented as mean values ±SEM. Statistical significance was determined by two-way ANOVA, numbers indicate P values. F: CFUs in the kidneys of WT and homozygous hHVCN1 mice infected intravenously with MUZ211 (CFU obtained from 11 WT and 24 hHVCN1 mice) and 62300D1 (CFU obtained from 11 WT and 10 hHVCN1 mice). Data for each isolate are from mice infected over three independent experiments and is represented as mean values ±SEM. Statistical significance was determined by t -test (two-tailed), numbers indicate P values. Also refer to Extended Data Figures 5 and 7 .

    Journal: Nature microbiology

    Article Title: Genetic variation of staphylococcal LukAB toxin determines receptor tropism

    doi: 10.1038/s41564-021-00890-3

    Figure Lengend Snippet: LukAB targeting of HVCN1 promotes S. aureus pathogenesis. A: Intoxication of murine PECs with indicated concentrations of leukocidins. Membrane damage was detected using Fixable Viability Dye eFluor ™ 450. Data are represented as the average of three independent experiments ± SEM B: ( inset ) Immunoblot of HVCN1 in CHO cells expressing firefly luciferase (Fluc), human (HVCN1) or murine (mHVCN1) HVCN1. Anti-actin immunoblot is shown above as a loading control. Representative images of four independent samples from one immunoblot are shown, see corresponding Source Data for full gel. Numbers on the left indicate migration of the corresponding molecular weight standards (in kDa). Target protein levels normalized by actin were obtained using ImageJ from four independent protein samples: HVCN1 = 0.310 ± 0.111, mHVCN1 = 0.333 ± 0.066 (mean ± SD), P = 0.742 as determined by unpaired t test. ( main figure ) Intoxication of Fluc, HVCN1, and mHVCN1 expressing CHO cells with indicated concentrations of CC30 LukAB. Cell viability was measured with Cell Titer. Data from three independent experiments are represented as mean values ±SD. Statistical significance was determined by two-way ANOVA (****, P ≤ 0.0001; ns, not significant). C: Schematic architecture of HVCN1 and the amino acid alignments of human and murine extracellular loops generated using Clustal Omega. D: Intoxication of Lenti-X 293T cells expressing C-terminal GFP-tagged human, murine, and chimeric HVCN1 proteins with indicated concentrations of CC30 LukAB. Membrane damage was detected using Fixable Viability Dye eFluor ™ 450. Data from three independent experiments are represented as the mean values ±SD. Statistical significance was determined by two-way ANOVA (****, P ≤ 0.0001; **, P ≤ 0.01; ns, not significant). E: Intoxication of PECs from wild type (WT) and hHVCN1 mice with indicated LukAB. Membrane damage was detected using Propidium Iodide (PI) incorporation. Data from five mice per genotype over three independent experiments are represented as mean values ±SEM. Statistical significance was determined by two-way ANOVA, numbers indicate P values. F: CFUs in the kidneys of WT and homozygous hHVCN1 mice infected intravenously with MUZ211 (CFU obtained from 11 WT and 24 hHVCN1 mice) and 62300D1 (CFU obtained from 11 WT and 10 hHVCN1 mice). Data for each isolate are from mice infected over three independent experiments and is represented as mean values ±SEM. Statistical significance was determined by t -test (two-tailed), numbers indicate P values. Also refer to Extended Data Figures 5 and 7 .

    Article Snippet: After blocking in 5% milk in PBS-T for 1 h at room temperature, membranes were incubated with primary antibodies ((anti-HVCN1 (1:200, AHC-001, Alomone Labs), and anti-β-Actin (1:1000, 715 8H10D10, Cell Signaling Technology)) diluted in 5% milk in PBS-T for 16 h at 4°C.

    Techniques: Expressing, Luciferase, Migration, Molecular Weight, Generated, Mouse Assay, Infection, Two Tailed Test