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Proteintech rabbit anti human integrin αv polyclonal antibody

a The graph displays the −log10-transformed RRA scores of genes enriched following infection with two SAFV-3 strains in HeLaN-∆SLC cells, analyzed using the MAGeCK software. The X -axis represents data from the screen using the SAFV-3 JPN08-356 strain, whereas the Y -axis shows results from the screen using the SAFV-3 JPN08-404 strain. The dotted line indicates the significance threshold of RRA = 0.01. Genes that met the criterion of RRA < 0.01 in both screens are highlighted in blue. The size of each dot reflects the combined enrichment across both screens, with larger dots indicating a greater sum of −log10 (RRA scores) from both experiments. b Expression of human <t>integrin</t> <t>αV</t> and integrin β8 in HeLaN-WT, HeLaN-∆AV, HeLaN-∆SLC∆AV, HeLaN-∆B8, and HeLaN-∆SLC∆B8 cells. The cells were stained with anti-integrin αV or anti-integrin αVβ8 antibodies and analyzed by flow cytometry. c HeLaN-WT, HeLaN-∆AV, HeLaN-∆B8, HeLaN-∆SLC∆AV, and HeLaN-∆SLC∆B8 cells were infected with tenfold serial dilutions of SAFV-3 and viable cells were stained with crystal violet to assess infection levels. Images are representative of two independent experiments. d Multi-step growth kinetics of SAFV-3 in HeLaN-∆B8, HeLaN-∆SLC∆B8, and HeLaN-WT cells. The cells were infected with SAFV-3 and incubated for up to 5 days. Data are presented as mean viral titers with s.d. ( n = 3). Statistical significance was determined using the two-sided Welch’s t -test. **, P < 0.01, *, P < 0.05, n.s. not significant. The dotted line indicates the limit of detection. Source data are provided as a Source Data file.

Rabbit Anti Human Integrin αv Polyclonal Antibody, supplied by Proteintech, used in various techniques. Bioz Stars score: 93/100, based on 28 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Saffold virus exploits integrin αvβ8 and sulfated glycosaminoglycans as cooperative attachment receptors for infection"

Article Title: Saffold virus exploits integrin αvβ8 and sulfated glycosaminoglycans as cooperative attachment receptors for infection

Journal: Nature Communications

doi: 10.1038/s41467-025-67236-z

Figure Legend Snippet: a The graph displays the −log10-transformed RRA scores of genes enriched following infection with two SAFV-3 strains in HeLaN-∆SLC cells, analyzed using the MAGeCK software. The X -axis represents data from the screen using the SAFV-3 JPN08-356 strain, whereas the Y -axis shows results from the screen using the SAFV-3 JPN08-404 strain. The dotted line indicates the significance threshold of RRA = 0.01. Genes that met the criterion of RRA < 0.01 in both screens are highlighted in blue. The size of each dot reflects the combined enrichment across both screens, with larger dots indicating a greater sum of −log10 (RRA scores) from both experiments. b Expression of human integrin αV and integrin β8 in HeLaN-WT, HeLaN-∆AV, HeLaN-∆SLC∆AV, HeLaN-∆B8, and HeLaN-∆SLC∆B8 cells. The cells were stained with anti-integrin αV or anti-integrin αVβ8 antibodies and analyzed by flow cytometry. c HeLaN-WT, HeLaN-∆AV, HeLaN-∆B8, HeLaN-∆SLC∆AV, and HeLaN-∆SLC∆B8 cells were infected with tenfold serial dilutions of SAFV-3 and viable cells were stained with crystal violet to assess infection levels. Images are representative of two independent experiments. d Multi-step growth kinetics of SAFV-3 in HeLaN-∆B8, HeLaN-∆SLC∆B8, and HeLaN-WT cells. The cells were infected with SAFV-3 and incubated for up to 5 days. Data are presented as mean viral titers with s.d. ( n = 3). Statistical significance was determined using the two-sided Welch’s t -test. **, P < 0.01, *, P < 0.05, n.s. not significant. The dotted line indicates the limit of detection. Source data are provided as a Source Data file.

Techniques Used: Transformation Assay, Infection, Software, Expressing, Staining, Flow Cytometry, Incubation

a Western blot analysis of integrin αV (left panel) and integrin β8 (right panel) expression in BHK-21 cells. BHK-21 cells that were lentivirally transduced with either human integrin αV (BHK + human AV) or hamster integrin β8 (BHK + hamster B8) were used as positive controls. The anti-integrin αV antibody cross-reacted with both human and hamster integrin αV. Actin served as the loading control. b Expression of HS, integrin αV, and β8 in BHK-21 derivatives. BHK-21 cells were stained with anti-HS antibody (upper left panel). BHK-21 cells stably expressing human integrin αV and/or β8 (BHK + human AV, BHK + human B8, BHK + human AVB8), as well as the control cells, were stained with anti-integrin αV or anti-integrin αVβ8 antibodies. The cells were analyzed by flow cytometry. c Susceptibility analysis using SAF/UnaG in BHK-21 cells expressing human integrin αV and/or β8. UnaG-positive cells (green, upper panel) and Hoechst-stained nuclei (blue, lower panel) were imaged at 16 h post-infection. Scale bar, 200 μm. The percentage of infected cells was determined by examining at least 1000 cells. Data are representative of two independent experiments. d One-step growth kinetics of SAFV-3 in BHK + human AV, BHK + human B8, BHK + human AVB8, and control cells. The cells were infected with SAFV-3 and incubated for up to 24 h. The dotted line indicates the limit of detection. e Western blot analysis of exogenous integrin β8 expression in BHK-21 cells lentivirally transduced with either mouse or hamster integrin β8. The anti-integrin β8 antibody cross-reacted with both mouse and hamster integrin β8. Actin served as the loading control. f Susceptibility analysis using SAF/UnaG in mouse and hamster integrin β8 expressing BHK-21 cells. UnaG-positive cells (green, upper panel) and Hoechst-stained nuclei (blue, lower panel) were captured at 16 h post-infection. Scale bar, 200 μm. Images are representative of two independent experiments. Data in ( c and d ) represent means with s.d. ( n = 3). Statistical significance was determined using a one-way ANOVA with Dunnett’s multiple comparison test ( c ) and the two-sided Welch’s t -test ( d ). **, P < 0.01, *, P < 0.05, n.s. not significant. Source data are provided as a Source Data file.

Figure Legend Snippet: a Western blot analysis of integrin αV (left panel) and integrin β8 (right panel) expression in BHK-21 cells. BHK-21 cells that were lentivirally transduced with either human integrin αV (BHK + human AV) or hamster integrin β8 (BHK + hamster B8) were used as positive controls. The anti-integrin αV antibody cross-reacted with both human and hamster integrin αV. Actin served as the loading control. b Expression of HS, integrin αV, and β8 in BHK-21 derivatives. BHK-21 cells were stained with anti-HS antibody (upper left panel). BHK-21 cells stably expressing human integrin αV and/or β8 (BHK + human AV, BHK + human B8, BHK + human AVB8), as well as the control cells, were stained with anti-integrin αV or anti-integrin αVβ8 antibodies. The cells were analyzed by flow cytometry. c Susceptibility analysis using SAF/UnaG in BHK-21 cells expressing human integrin αV and/or β8. UnaG-positive cells (green, upper panel) and Hoechst-stained nuclei (blue, lower panel) were imaged at 16 h post-infection. Scale bar, 200 μm. The percentage of infected cells was determined by examining at least 1000 cells. Data are representative of two independent experiments. d One-step growth kinetics of SAFV-3 in BHK + human AV, BHK + human B8, BHK + human AVB8, and control cells. The cells were infected with SAFV-3 and incubated for up to 24 h. The dotted line indicates the limit of detection. e Western blot analysis of exogenous integrin β8 expression in BHK-21 cells lentivirally transduced with either mouse or hamster integrin β8. The anti-integrin β8 antibody cross-reacted with both mouse and hamster integrin β8. Actin served as the loading control. f Susceptibility analysis using SAF/UnaG in mouse and hamster integrin β8 expressing BHK-21 cells. UnaG-positive cells (green, upper panel) and Hoechst-stained nuclei (blue, lower panel) were captured at 16 h post-infection. Scale bar, 200 μm. Images are representative of two independent experiments. Data in ( c and d ) represent means with s.d. ( n = 3). Statistical significance was determined using a one-way ANOVA with Dunnett’s multiple comparison test ( c ) and the two-sided Welch’s t -test ( d ). **, P < 0.01, *, P < 0.05, n.s. not significant. Source data are provided as a Source Data file.

Techniques Used: Western Blot, Expressing, Transduction, Control, Staining, Stable Transfection, Flow Cytometry, Infection, Incubation, Comparison

a Expression of human integrin β subunits (β1, β3, β5, and β6) on the surface of BHK-21 derivatives. BHK-21 cells lentivirally transduced with the respective integrin β subunits were stained with the indicated antibodies and analyzed using flow cytometry. b Susceptibility analysis using SAF/UnaG in human integrin αV and the indicated β subunit expressing BHK-21 cells. UnaG-positive cells (green, upper panel) and nuclei stained with Hoechst (blue, lower panel) were imaged at 16 h post-infection. Scale bar, 200 μm. Images are representative of two independent experiments.

Figure Legend Snippet: a Expression of human integrin β subunits (β1, β3, β5, and β6) on the surface of BHK-21 derivatives. BHK-21 cells lentivirally transduced with the respective integrin β subunits were stained with the indicated antibodies and analyzed using flow cytometry. b Susceptibility analysis using SAF/UnaG in human integrin αV and the indicated β subunit expressing BHK-21 cells. UnaG-positive cells (green, upper panel) and nuclei stained with Hoechst (blue, lower panel) were imaged at 16 h post-infection. Scale bar, 200 μm. Images are representative of two independent experiments.

Techniques Used: Expressing, Transduction, Staining, Flow Cytometry, Infection

a Pull-down assay of SAFV-3 using heparin (left panel) and integrin αVβ8 (right panel). Heparin and Fc chimera of extracellular domains of integrin αVβ8, αVβ3, or the signal sequence (ss) of integrin αV (negative control) were prepared as complexes with magnetic beads. These complexes were incubated with SAFV-3, followed by western blot analysis of the bound virus using anti-SAFV-3 antiserum (left and right upper panels). The bottom right panel shows an image of the integrin-Fc complex on magnetic beads used for pulldown, detected using an anti-mouse IgG antibody. b Cell surface attachment assay for SAFV-3. HeLaN-WT, HeLaN-∆SLC, HeLaN-∆B8, HeLaN-∆SLC∆B8, and HeLaN-∆SLC + human AVB8 were incubated with SAFV-3, followed by RT-qPCR analysis of the bound virus. c Expression of human integrin β8 in HeLaN-∆SLC and HeLaN-∆SLC + human AVB8 cells. To compare the expression levels of integrin αVβ8 on the cell surface, HeLaN-∆SLC and HeLaN-∆SLC + human AVB8 cells were stained with anti-integrin αVβ8 antibodies and analyzed by flow cytometry. d , e Inhibition of SAFV-3 attachment to the cell surface by soluble heparin ( d ) or recombinant integrin αVβ8 ( e ). HeLaN-WT cells ( d ) or HeLaN-∆SLC + human AVB8 cells ( e ) were incubated with SAFV-3 pretreated with 1 or 10 μg of soluble heparin or recombinant integrin αVβ8, respectively. Recombinant integrin αVβ3 was the negative control. After incubation at 4 °C for 2 h, bound virus was analyzed using RT-qPCR. f HeLaN-WT, HeLaN-∆SLC, HeLaN-∆B8, HeLaN-∆SLC∆B8, and HeLaN-∆SLC + human AVB8 cells were infected with tenfold serial dilutions of SAFV-3, and viable cells were stained with crystal violet to assess infection levels. All data are representative of two independent experiments. Data in ( b , d , and e ) represent means with s.d. ( n = 3). Statistical significance was determined using a one-way ANOVA with Dunnett’s multiple comparison test. **, P < 0.01, n.s. not significant. Asterisks directly placed on bars indicate statistically significant differences compared to WT or untreated samples, while asterisks placed on the lines connecting bars denote statistically significant differences between those bars. Source data are provided as a Source Data file. Ag antigen, Fc fragment crystallizable region.

Figure Legend Snippet: a Pull-down assay of SAFV-3 using heparin (left panel) and integrin αVβ8 (right panel). Heparin and Fc chimera of extracellular domains of integrin αVβ8, αVβ3, or the signal sequence (ss) of integrin αV (negative control) were prepared as complexes with magnetic beads. These complexes were incubated with SAFV-3, followed by western blot analysis of the bound virus using anti-SAFV-3 antiserum (left and right upper panels). The bottom right panel shows an image of the integrin-Fc complex on magnetic beads used for pulldown, detected using an anti-mouse IgG antibody. b Cell surface attachment assay for SAFV-3. HeLaN-WT, HeLaN-∆SLC, HeLaN-∆B8, HeLaN-∆SLC∆B8, and HeLaN-∆SLC + human AVB8 were incubated with SAFV-3, followed by RT-qPCR analysis of the bound virus. c Expression of human integrin β8 in HeLaN-∆SLC and HeLaN-∆SLC + human AVB8 cells. To compare the expression levels of integrin αVβ8 on the cell surface, HeLaN-∆SLC and HeLaN-∆SLC + human AVB8 cells were stained with anti-integrin αVβ8 antibodies and analyzed by flow cytometry. d , e Inhibition of SAFV-3 attachment to the cell surface by soluble heparin ( d ) or recombinant integrin αVβ8 ( e ). HeLaN-WT cells ( d ) or HeLaN-∆SLC + human AVB8 cells ( e ) were incubated with SAFV-3 pretreated with 1 or 10 μg of soluble heparin or recombinant integrin αVβ8, respectively. Recombinant integrin αVβ3 was the negative control. After incubation at 4 °C for 2 h, bound virus was analyzed using RT-qPCR. f HeLaN-WT, HeLaN-∆SLC, HeLaN-∆B8, HeLaN-∆SLC∆B8, and HeLaN-∆SLC + human AVB8 cells were infected with tenfold serial dilutions of SAFV-3, and viable cells were stained with crystal violet to assess infection levels. All data are representative of two independent experiments. Data in ( b , d , and e ) represent means with s.d. ( n = 3). Statistical significance was determined using a one-way ANOVA with Dunnett’s multiple comparison test. **, P < 0.01, n.s. not significant. Asterisks directly placed on bars indicate statistically significant differences compared to WT or untreated samples, while asterisks placed on the lines connecting bars denote statistically significant differences between those bars. Source data are provided as a Source Data file. Ag antigen, Fc fragment crystallizable region.

Techniques Used: Pull Down Assay, Sequencing, Negative Control, Magnetic Beads, Incubation, Western Blot, Virus, Quantitative RT-PCR, Expressing, Staining, Flow Cytometry, Inhibition, Recombinant, Infection, Comparison

a Expression of HS and human integrin β8 in BHK-WT, BHK-∆SLC, BHK + human AVB8, BHK-∆SLC + human AVB8, and revertant cells expressing human SLC35B2 (BHK-∆SLC + human AVB8 + SLC). The cells were stained with anti-HS or anti-integrin αVβ8 antibodies and analyzed by flow cytometry. b Cell surface attachment assay for SAFV-3. BHK-WT and BHK-∆SLC cells were incubated with SAFV-3 at 4 °C for 2 h. Viral binding to HS was assessed by RT-qPCR quantification of cell-bound virus ( n = 3). c Susceptibility analysis using SAF/UnaG in BHK + human AVB8, BHK-∆SLC + human AVB8, and BHK-∆SLC + human AVB8 + SLC cells. The cells used in this experiment were sorted to equalize the surface expression levels of integrin αVβ8 between BHK + human AVB8 and BHK-∆SLC + human AVB8 cells. UnaG-positive cells (green) and nuclei stained with Hoechst (blue) were imaged at 16 h post-infection. The percentage of infected cells was determined by examining at least 800 cells/well ( n = 4). Scale bar, 200 μm. d Cell surface attachment assay for SAFV-3 in BHK + human AVB8, BHK-∆SLC + human AVB8, and BHK-∆SLC + human AVB8 + SLC cells ( n = 3). Bar graphs in ( b– d ) are presented as means with s.d. Statistical significance was determined using the two-sided Welch’s t -test ( b ) and a one-way ANOVA with Dunnett’s multiple comparison test ( c , d ). **, P < 0.01, *, P < 0.05. All data are representative of two independent experiments. Source data are provided as a Source Data file.

Figure Legend Snippet: a Expression of HS and human integrin β8 in BHK-WT, BHK-∆SLC, BHK + human AVB8, BHK-∆SLC + human AVB8, and revertant cells expressing human SLC35B2 (BHK-∆SLC + human AVB8 + SLC). The cells were stained with anti-HS or anti-integrin αVβ8 antibodies and analyzed by flow cytometry. b Cell surface attachment assay for SAFV-3. BHK-WT and BHK-∆SLC cells were incubated with SAFV-3 at 4 °C for 2 h. Viral binding to HS was assessed by RT-qPCR quantification of cell-bound virus ( n = 3). c Susceptibility analysis using SAF/UnaG in BHK + human AVB8, BHK-∆SLC + human AVB8, and BHK-∆SLC + human AVB8 + SLC cells. The cells used in this experiment were sorted to equalize the surface expression levels of integrin αVβ8 between BHK + human AVB8 and BHK-∆SLC + human AVB8 cells. UnaG-positive cells (green) and nuclei stained with Hoechst (blue) were imaged at 16 h post-infection. The percentage of infected cells was determined by examining at least 800 cells/well ( n = 4). Scale bar, 200 μm. d Cell surface attachment assay for SAFV-3 in BHK + human AVB8, BHK-∆SLC + human AVB8, and BHK-∆SLC + human AVB8 + SLC cells ( n = 3). Bar graphs in ( b– d ) are presented as means with s.d. Statistical significance was determined using the two-sided Welch’s t -test ( b ) and a one-way ANOVA with Dunnett’s multiple comparison test ( c , d ). **, P < 0.01, *, P < 0.05. All data are representative of two independent experiments. Source data are provided as a Source Data file.

Techniques Used: Expressing, Staining, Flow Cytometry, Incubation, Binding Assay, Quantitative RT-PCR, Virus, Infection, Comparison

a Viral infection analysis using integrin β8 mutants. BHK-21 cells expressing the human integrin β8 mutants (∆SDL, Y172N, and I208R) were inoculated with SAFV-3. After 2 days, virus titers were determined using the TCID 50 assay. Human integrin β3 was the negative control. The dotted line indicates the limit of detection. The right panel shows the results of western blot analysis of integrin β8 mutants and integrin β3 expression in BHK-21 cells. b Alignment of the amino acid sequences of puff A on VP2 of SAFV-3 (left panel) and CD loop I on VP1 of SAFV-2 (right panel). RGD-like sequences are highlighted. c Infection blocking assay using RGD peptide. Left panel: HeLaN-∆SLC cells were pretreated with 10 or 100 μg of GRGDS, GRADS, or GRAES peptide at 4 °C for 30 min and then incubated with SAFV-3/UnaG virus for an additional 60 min. Right panel: HeLaN-∆SLC cells were pretreated with 10 or 100 μg of GRGDS, GRLDS, or GRAES peptide for 30 min and then incubated with SAFV-2/UnaG virus for an additional 60 min. GRGDS and GRAES peptides were positive and negative controls, respectively. The number of UnaG-positive cells at 14 h post-infection was counted using ImageJ software. d Schematic illustration of mutagenesis in RGD-like sequence. The mutated amino acid residues are highlighted. e HeLaN-∆SLC∆B8, HeLaN-∆SLC, and HeLaN-∆SLC + human AVB8 cells were infected with tenfold serial dilutions of mutant viruses carrying mutations in the RGD-like sequences, and viable cells were stained with crystal violet. Primary progeny virus produced from BHK cells transfected with infectious RNA (P-0 virus) was used. Bar graphs in ( a and c ) represent means with s.d. ( n = 3 in ( a ); n = 9 peptide-free and n = 3 peptide-added samples in ( c )). Statistical significance was determined using a one-way ANOVA with Dunnett’s multiple comparison test. **, P < 0.01, n.s. not significant. Statistical comparisons in ( c ) were made between peptide-free and peptide-added samples. All data are representative of two independent experiments. Source data are provided as a Source Data file.

Figure Legend Snippet: a Viral infection analysis using integrin β8 mutants. BHK-21 cells expressing the human integrin β8 mutants (∆SDL, Y172N, and I208R) were inoculated with SAFV-3. After 2 days, virus titers were determined using the TCID 50 assay. Human integrin β3 was the negative control. The dotted line indicates the limit of detection. The right panel shows the results of western blot analysis of integrin β8 mutants and integrin β3 expression in BHK-21 cells. b Alignment of the amino acid sequences of puff A on VP2 of SAFV-3 (left panel) and CD loop I on VP1 of SAFV-2 (right panel). RGD-like sequences are highlighted. c Infection blocking assay using RGD peptide. Left panel: HeLaN-∆SLC cells were pretreated with 10 or 100 μg of GRGDS, GRADS, or GRAES peptide at 4 °C for 30 min and then incubated with SAFV-3/UnaG virus for an additional 60 min. Right panel: HeLaN-∆SLC cells were pretreated with 10 or 100 μg of GRGDS, GRLDS, or GRAES peptide for 30 min and then incubated with SAFV-2/UnaG virus for an additional 60 min. GRGDS and GRAES peptides were positive and negative controls, respectively. The number of UnaG-positive cells at 14 h post-infection was counted using ImageJ software. d Schematic illustration of mutagenesis in RGD-like sequence. The mutated amino acid residues are highlighted. e HeLaN-∆SLC∆B8, HeLaN-∆SLC, and HeLaN-∆SLC + human AVB8 cells were infected with tenfold serial dilutions of mutant viruses carrying mutations in the RGD-like sequences, and viable cells were stained with crystal violet. Primary progeny virus produced from BHK cells transfected with infectious RNA (P-0 virus) was used. Bar graphs in ( a and c ) represent means with s.d. ( n = 3 in ( a ); n = 9 peptide-free and n = 3 peptide-added samples in ( c )). Statistical significance was determined using a one-way ANOVA with Dunnett’s multiple comparison test. **, P < 0.01, n.s. not significant. Statistical comparisons in ( c ) were made between peptide-free and peptide-added samples. All data are representative of two independent experiments. Source data are provided as a Source Data file.

Techniques Used: Infection, Expressing, Virus, Negative Control, Western Blot, Blocking Assay, Incubation, Software, Mutagenesis, Sequencing, Staining, Produced, Transfection, Comparison

Sulfated GAGs and integrin αVβ8 function as interconnected dual receptors for SAFV infection in HeLa-N cells. SAFV can directly bind to either sulfated GAGs or integrin αVβ8, while a portion of viruses bound to sulfated GAGs subsequently interact with integrin αVβ8. In addition, the data suggest the existence of a downstream molecule (factor X) required for an unspecified step in the viral entry process following sulfated GAGs binding.

Figure Legend Snippet: Sulfated GAGs and integrin αVβ8 function as interconnected dual receptors for SAFV infection in HeLa-N cells. SAFV can directly bind to either sulfated GAGs or integrin αVβ8, while a portion of viruses bound to sulfated GAGs subsequently interact with integrin αVβ8. In addition, the data suggest the existence of a downstream molecule (factor X) required for an unspecified step in the viral entry process following sulfated GAGs binding.

Techniques Used: Infection, Binding Assay

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Journal: Nature Communications

Article Title: Saffold virus exploits integrin αvβ8 and sulfated glycosaminoglycans as cooperative attachment receptors for infection

doi: 10.1038/s41467-025-67236-z

Figure Lengend Snippet: a The graph displays the −log10-transformed RRA scores of genes enriched following infection with two SAFV-3 strains in HeLaN-∆SLC cells, analyzed using the MAGeCK software. The X -axis represents data from the screen using the SAFV-3 JPN08-356 strain, whereas the Y -axis shows results from the screen using the SAFV-3 JPN08-404 strain. The dotted line indicates the significance threshold of RRA = 0.01. Genes that met the criterion of RRA < 0.01 in both screens are highlighted in blue. The size of each dot reflects the combined enrichment across both screens, with larger dots indicating a greater sum of −log10 (RRA scores) from both experiments. b Expression of human integrin αV and integrin β8 in HeLaN-WT, HeLaN-∆AV, HeLaN-∆SLC∆AV, HeLaN-∆B8, and HeLaN-∆SLC∆B8 cells. The cells were stained with anti-integrin αV or anti-integrin αVβ8 antibodies and analyzed by flow cytometry. c HeLaN-WT, HeLaN-∆AV, HeLaN-∆B8, HeLaN-∆SLC∆AV, and HeLaN-∆SLC∆B8 cells were infected with tenfold serial dilutions of SAFV-3 and viable cells were stained with crystal violet to assess infection levels. Images are representative of two independent experiments. d Multi-step growth kinetics of SAFV-3 in HeLaN-∆B8, HeLaN-∆SLC∆B8, and HeLaN-WT cells. The cells were infected with SAFV-3 and incubated for up to 5 days. Data are presented as mean viral titers with s.d. ( n = 3). Statistical significance was determined using the two-sided Welch’s t -test. **, P < 0.01, *, P < 0.05, n.s. not significant. The dotted line indicates the limit of detection. Source data are provided as a Source Data file.

Article Snippet: To detect of endogenous hamster integrin αV and β8, rabbit anti-human integrin αV polyclonal antibody (27096-1-AP, Proteintech) and rabbit anti-mouse integrin β8 (D1V7M) monoclonal antibody (88300, Cell Signaling Technology) were used, respectively.

Techniques: Transformation Assay, Infection, Software, Expressing, Staining, Flow Cytometry, Incubation

Journal: Nature Communications

Article Title: Saffold virus exploits integrin αvβ8 and sulfated glycosaminoglycans as cooperative attachment receptors for infection

doi: 10.1038/s41467-025-67236-z

Figure Lengend Snippet: a Western blot analysis of integrin αV (left panel) and integrin β8 (right panel) expression in BHK-21 cells. BHK-21 cells that were lentivirally transduced with either human integrin αV (BHK + human AV) or hamster integrin β8 (BHK + hamster B8) were used as positive controls. The anti-integrin αV antibody cross-reacted with both human and hamster integrin αV. Actin served as the loading control. b Expression of HS, integrin αV, and β8 in BHK-21 derivatives. BHK-21 cells were stained with anti-HS antibody (upper left panel). BHK-21 cells stably expressing human integrin αV and/or β8 (BHK + human AV, BHK + human B8, BHK + human AVB8), as well as the control cells, were stained with anti-integrin αV or anti-integrin αVβ8 antibodies. The cells were analyzed by flow cytometry. c Susceptibility analysis using SAF/UnaG in BHK-21 cells expressing human integrin αV and/or β8. UnaG-positive cells (green, upper panel) and Hoechst-stained nuclei (blue, lower panel) were imaged at 16 h post-infection. Scale bar, 200 μm. The percentage of infected cells was determined by examining at least 1000 cells. Data are representative of two independent experiments. d One-step growth kinetics of SAFV-3 in BHK + human AV, BHK + human B8, BHK + human AVB8, and control cells. The cells were infected with SAFV-3 and incubated for up to 24 h. The dotted line indicates the limit of detection. e Western blot analysis of exogenous integrin β8 expression in BHK-21 cells lentivirally transduced with either mouse or hamster integrin β8. The anti-integrin β8 antibody cross-reacted with both mouse and hamster integrin β8. Actin served as the loading control. f Susceptibility analysis using SAF/UnaG in mouse and hamster integrin β8 expressing BHK-21 cells. UnaG-positive cells (green, upper panel) and Hoechst-stained nuclei (blue, lower panel) were captured at 16 h post-infection. Scale bar, 200 μm. Images are representative of two independent experiments. Data in ( c and d ) represent means with s.d. ( n = 3). Statistical significance was determined using a one-way ANOVA with Dunnett’s multiple comparison test ( c ) and the two-sided Welch’s t -test ( d ). **, P < 0.01, *, P < 0.05, n.s. not significant. Source data are provided as a Source Data file.

Techniques: Western Blot, Expressing, Transduction, Control, Staining, Stable Transfection, Flow Cytometry, Infection, Incubation, Comparison

Journal: Nature Communications

Article Title: Saffold virus exploits integrin αvβ8 and sulfated glycosaminoglycans as cooperative attachment receptors for infection

doi: 10.1038/s41467-025-67236-z

Figure Lengend Snippet: a Expression of human integrin β subunits (β1, β3, β5, and β6) on the surface of BHK-21 derivatives. BHK-21 cells lentivirally transduced with the respective integrin β subunits were stained with the indicated antibodies and analyzed using flow cytometry. b Susceptibility analysis using SAF/UnaG in human integrin αV and the indicated β subunit expressing BHK-21 cells. UnaG-positive cells (green, upper panel) and nuclei stained with Hoechst (blue, lower panel) were imaged at 16 h post-infection. Scale bar, 200 μm. Images are representative of two independent experiments.

Techniques: Expressing, Transduction, Staining, Flow Cytometry, Infection

Journal: Nature Communications

Article Title: Saffold virus exploits integrin αvβ8 and sulfated glycosaminoglycans as cooperative attachment receptors for infection

doi: 10.1038/s41467-025-67236-z

Figure Lengend Snippet: a Pull-down assay of SAFV-3 using heparin (left panel) and integrin αVβ8 (right panel). Heparin and Fc chimera of extracellular domains of integrin αVβ8, αVβ3, or the signal sequence (ss) of integrin αV (negative control) were prepared as complexes with magnetic beads. These complexes were incubated with SAFV-3, followed by western blot analysis of the bound virus using anti-SAFV-3 antiserum (left and right upper panels). The bottom right panel shows an image of the integrin-Fc complex on magnetic beads used for pulldown, detected using an anti-mouse IgG antibody. b Cell surface attachment assay for SAFV-3. HeLaN-WT, HeLaN-∆SLC, HeLaN-∆B8, HeLaN-∆SLC∆B8, and HeLaN-∆SLC + human AVB8 were incubated with SAFV-3, followed by RT-qPCR analysis of the bound virus. c Expression of human integrin β8 in HeLaN-∆SLC and HeLaN-∆SLC + human AVB8 cells. To compare the expression levels of integrin αVβ8 on the cell surface, HeLaN-∆SLC and HeLaN-∆SLC + human AVB8 cells were stained with anti-integrin αVβ8 antibodies and analyzed by flow cytometry. d , e Inhibition of SAFV-3 attachment to the cell surface by soluble heparin ( d ) or recombinant integrin αVβ8 ( e ). HeLaN-WT cells ( d ) or HeLaN-∆SLC + human AVB8 cells ( e ) were incubated with SAFV-3 pretreated with 1 or 10 μg of soluble heparin or recombinant integrin αVβ8, respectively. Recombinant integrin αVβ3 was the negative control. After incubation at 4 °C for 2 h, bound virus was analyzed using RT-qPCR. f HeLaN-WT, HeLaN-∆SLC, HeLaN-∆B8, HeLaN-∆SLC∆B8, and HeLaN-∆SLC + human AVB8 cells were infected with tenfold serial dilutions of SAFV-3, and viable cells were stained with crystal violet to assess infection levels. All data are representative of two independent experiments. Data in ( b , d , and e ) represent means with s.d. ( n = 3). Statistical significance was determined using a one-way ANOVA with Dunnett’s multiple comparison test. **, P < 0.01, n.s. not significant. Asterisks directly placed on bars indicate statistically significant differences compared to WT or untreated samples, while asterisks placed on the lines connecting bars denote statistically significant differences between those bars. Source data are provided as a Source Data file. Ag antigen, Fc fragment crystallizable region.

Techniques: Pull Down Assay, Sequencing, Negative Control, Magnetic Beads, Incubation, Western Blot, Virus, Quantitative RT-PCR, Expressing, Staining, Flow Cytometry, Inhibition, Recombinant, Infection, Comparison

Journal: Nature Communications

Article Title: Saffold virus exploits integrin αvβ8 and sulfated glycosaminoglycans as cooperative attachment receptors for infection

doi: 10.1038/s41467-025-67236-z

Figure Lengend Snippet: a Expression of HS and human integrin β8 in BHK-WT, BHK-∆SLC, BHK + human AVB8, BHK-∆SLC + human AVB8, and revertant cells expressing human SLC35B2 (BHK-∆SLC + human AVB8 + SLC). The cells were stained with anti-HS or anti-integrin αVβ8 antibodies and analyzed by flow cytometry. b Cell surface attachment assay for SAFV-3. BHK-WT and BHK-∆SLC cells were incubated with SAFV-3 at 4 °C for 2 h. Viral binding to HS was assessed by RT-qPCR quantification of cell-bound virus ( n = 3). c Susceptibility analysis using SAF/UnaG in BHK + human AVB8, BHK-∆SLC + human AVB8, and BHK-∆SLC + human AVB8 + SLC cells. The cells used in this experiment were sorted to equalize the surface expression levels of integrin αVβ8 between BHK + human AVB8 and BHK-∆SLC + human AVB8 cells. UnaG-positive cells (green) and nuclei stained with Hoechst (blue) were imaged at 16 h post-infection. The percentage of infected cells was determined by examining at least 800 cells/well ( n = 4). Scale bar, 200 μm. d Cell surface attachment assay for SAFV-3 in BHK + human AVB8, BHK-∆SLC + human AVB8, and BHK-∆SLC + human AVB8 + SLC cells ( n = 3). Bar graphs in ( b– d ) are presented as means with s.d. Statistical significance was determined using the two-sided Welch’s t -test ( b ) and a one-way ANOVA with Dunnett’s multiple comparison test ( c , d ). **, P < 0.01, *, P < 0.05. All data are representative of two independent experiments. Source data are provided as a Source Data file.

Techniques: Expressing, Staining, Flow Cytometry, Incubation, Binding Assay, Quantitative RT-PCR, Virus, Infection, Comparison

Journal: Nature Communications

Article Title: Saffold virus exploits integrin αvβ8 and sulfated glycosaminoglycans as cooperative attachment receptors for infection

doi: 10.1038/s41467-025-67236-z

Figure Lengend Snippet: a Viral infection analysis using integrin β8 mutants. BHK-21 cells expressing the human integrin β8 mutants (∆SDL, Y172N, and I208R) were inoculated with SAFV-3. After 2 days, virus titers were determined using the TCID 50 assay. Human integrin β3 was the negative control. The dotted line indicates the limit of detection. The right panel shows the results of western blot analysis of integrin β8 mutants and integrin β3 expression in BHK-21 cells. b Alignment of the amino acid sequences of puff A on VP2 of SAFV-3 (left panel) and CD loop I on VP1 of SAFV-2 (right panel). RGD-like sequences are highlighted. c Infection blocking assay using RGD peptide. Left panel: HeLaN-∆SLC cells were pretreated with 10 or 100 μg of GRGDS, GRADS, or GRAES peptide at 4 °C for 30 min and then incubated with SAFV-3/UnaG virus for an additional 60 min. Right panel: HeLaN-∆SLC cells were pretreated with 10 or 100 μg of GRGDS, GRLDS, or GRAES peptide for 30 min and then incubated with SAFV-2/UnaG virus for an additional 60 min. GRGDS and GRAES peptides were positive and negative controls, respectively. The number of UnaG-positive cells at 14 h post-infection was counted using ImageJ software. d Schematic illustration of mutagenesis in RGD-like sequence. The mutated amino acid residues are highlighted. e HeLaN-∆SLC∆B8, HeLaN-∆SLC, and HeLaN-∆SLC + human AVB8 cells were infected with tenfold serial dilutions of mutant viruses carrying mutations in the RGD-like sequences, and viable cells were stained with crystal violet. Primary progeny virus produced from BHK cells transfected with infectious RNA (P-0 virus) was used. Bar graphs in ( a and c ) represent means with s.d. ( n = 3 in ( a ); n = 9 peptide-free and n = 3 peptide-added samples in ( c )). Statistical significance was determined using a one-way ANOVA with Dunnett’s multiple comparison test. **, P < 0.01, n.s. not significant. Statistical comparisons in ( c ) were made between peptide-free and peptide-added samples. All data are representative of two independent experiments. Source data are provided as a Source Data file.

Techniques: Infection, Expressing, Virus, Negative Control, Western Blot, Blocking Assay, Incubation, Software, Mutagenesis, Sequencing, Staining, Produced, Transfection, Comparison

Journal: Nature Communications

Article Title: Saffold virus exploits integrin αvβ8 and sulfated glycosaminoglycans as cooperative attachment receptors for infection

doi: 10.1038/s41467-025-67236-z

Figure Lengend Snippet: Sulfated GAGs and integrin αVβ8 function as interconnected dual receptors for SAFV infection in HeLa-N cells. SAFV can directly bind to either sulfated GAGs or integrin αVβ8, while a portion of viruses bound to sulfated GAGs subsequently interact with integrin αVβ8. In addition, the data suggest the existence of a downstream molecule (factor X) required for an unspecified step in the viral entry process following sulfated GAGs binding.

Techniques: Infection, Binding Assay

Fig. 5. Biological evaluation of PDC 4d. (A) Chemical structure of PDC 4d. (B) Western blot analysis of integrin αv expression in HEK-293, U87MG, and A549 cells. (C) Quantitative analysis of integrin αv expression levels in HEK-293, U87MG, and A549 cells. Statistical significance was determined by one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test. ****P < 0.0001. (D) Cytotoxicity evaluation of PDC 4d, c(RGDfM), and PTX in HEK-293, U87MG, and A549 cells, respec- tively. Data are shown as means ± SEM (n = 3 biological replicates).

Journal: Science advances

Article Title: Controlled reversible methionine-selective sulfimidation of peptides.

doi: 10.1126/sciadv.adv8712

Figure Lengend Snippet: Fig. 5. Biological evaluation of PDC 4d. (A) Chemical structure of PDC 4d. (B) Western blot analysis of integrin αv expression in HEK-293, U87MG, and A549 cells. (C) Quantitative analysis of integrin αv expression levels in HEK-293, U87MG, and A549 cells. Statistical significance was determined by one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test. ****P < 0.0001. (D) Cytotoxicity evaluation of PDC 4d, c(RGDfM), and PTX in HEK-293, U87MG, and A549 cells, respec- tively. Data are shown as means ± SEM (n = 3 biological replicates).

Article Snippet: Integrin αv polyclonal antibody (27096- 1- AP) and β actin polyclonal antibody (20536- 1- AP) were purchased from Proteintech; horseradish peroxidase–labeled Goat Anti- Rabbit IgG(H+L) (A0208) was purchased from Beyotime Biotechnology.

Techniques: Western Blot, Expressing

Differentiation of ER-Hoxb8 cells into osteoclasts is accompanied by morphologic changes, increased transcription of marker genes, lacuna formation, and distinct localization of integrin α2β1. (A) ER-Hoxb8 cells seeded onto glass coverslips differentiated in response to M-CSF and RANKL. Differential interference contrast images taken with a laser scanning microscope at 10× magnification on the indicated days of differentiation (at least 3 independent experiments). (B) At different time points, mRNA was isolated from differentiating ER-Hoxb8 cells, and transcripts of ITGA2, ITGAV, RANK and TRAP were quantified by qRT-PCR (data are mean ± S.E.M. from 10 independent experiments. (C) Histochemical staining of TRAP in the resorption lacuna and transcytotic vesicles of ER-Hoxb8-derived osteoclasts on day 7 of differentiation on glass (at least 3 independent experiments). (D) Confocal images of ER-Hoxb8 cells differentiated on glass coverslips for 7 days and stained for integrin α2 subunit (green), F-actin (red) and nuclei (blue). (E) Enlarged details of regions of interests indicated in (D) : Integrin α2β1 in i) tunneling nanotubes, ii) podosomes near the cell periphery, and iii) at the sealing zone of a resorption lacuna. (F) Integrin α2 in filopodia. Representative images of n = 4 independent experiments are shown.

Journal: Frontiers in Cell and Developmental Biology

Article Title: Integrin α2 is an early marker for osteoclast differentiation that contributes to key steps in osteoclastogenesis

doi: 10.3389/fcell.2024.1448725

Figure Lengend Snippet: Differentiation of ER-Hoxb8 cells into osteoclasts is accompanied by morphologic changes, increased transcription of marker genes, lacuna formation, and distinct localization of integrin α2β1. (A) ER-Hoxb8 cells seeded onto glass coverslips differentiated in response to M-CSF and RANKL. Differential interference contrast images taken with a laser scanning microscope at 10× magnification on the indicated days of differentiation (at least 3 independent experiments). (B) At different time points, mRNA was isolated from differentiating ER-Hoxb8 cells, and transcripts of ITGA2, ITGAV, RANK and TRAP were quantified by qRT-PCR (data are mean ± S.E.M. from 10 independent experiments. (C) Histochemical staining of TRAP in the resorption lacuna and transcytotic vesicles of ER-Hoxb8-derived osteoclasts on day 7 of differentiation on glass (at least 3 independent experiments). (D) Confocal images of ER-Hoxb8 cells differentiated on glass coverslips for 7 days and stained for integrin α2 subunit (green), F-actin (red) and nuclei (blue). (E) Enlarged details of regions of interests indicated in (D) : Integrin α2β1 in i) tunneling nanotubes, ii) podosomes near the cell periphery, and iii) at the sealing zone of a resorption lacuna. (F) Integrin α2 in filopodia. Representative images of n = 4 independent experiments are shown.

Article Snippet: After permeabilization with 0.1% Triton-X100 or 0.1% saponin in PBS, samples were blocked with 5% normal serum matching the secondary antibody and 1% BSA in PBS for 1 h. Subsequently, they were incubated overnight at 4°C with primary antibodies against integrin α2 (1:500, monoclonal rabbit NBP2-67691, Bio-Techne, Abington, United Kingdom), integrin αV (1:100, polyclonal rabbit antibodies, Bioss bs-1310R, Woburn, MA, United States), DC-STAMP (1:50, monoclonal mouse MABF39-I clone 1A2, Sigma-Aldrich), and CD9 (1:500, monoclonal rat MA1-10309, Invitrogen, Waltham, MA, United States) in blocking solution.

Techniques: Marker, Laser-Scanning Microscopy, Isolation, Quantitative RT-PCR, Staining, Derivative Assay

Integrin α2β1 deficiency cannot be compensated by OSCAR and results in reduced osteoclastic syncytia formation and hydroxyapatite resorption. (A) qRT-PCR of OSCAR at different time points of differentiation of ER-Hoxb8 ITGA2wt and -ITGA2-KO cells on collagen-I-coated or uncoated glass coverslips (n = 6 and 8, respectively). (B) Quantification of cell fusion after 7 days of differentiation via the number of Hoechst 33342-stained nuclei per cell. More than 7,500 cells in 3 independent experiments were analyzed. A Pearson Chi-squared test revealed a significant difference between the ITGA2wt and ITGA2-KO cell populations ( p < 0.0001), but no significant difference for each of the 2 cell variants between collagen-I and uncoated glass. (C) qRT-PCR of DC-STAMP at different time points of differentiation of Hoxb8-ITGA2wt and -ITGA2-KO cells on collagen-I-coated or uncoated glass coverslips. (D) Resorption areas (light grey) of ER-Hoxb8-derived osteoclasts after 10 days of differentiation on hydroxyapatite-coated coverslips. Cells were removed and remaining hydroxyapatite was silver-stained. Representative images of 8 independent experiments are shown. (E) Biometric quantification of resorption areas in (D) using the trainable WEKA segmentation of Fiji (ImageJ 1.53t), n = 8 ± S.E.M., p < 0.05. (F) qRT-PCR of TRAP at different time points of differentiation of ITGA2wt and ITGA2-KO ER-Hoxb8 cells on collagen-I-coated or uncoated glass coverslips. (G) qRT-PCR of CTSK at different time points of differentiation of ITGA2wt and -ITGA2-KO cells on collagen-I-coated or uncoated glass coverslips. qRT-PCR data (panels C , F , and G ) are represented as mean ± S.E.M. from three to four independent experiments in duplicate with Student’s t-test showing pairwise significances as p < 0.05 (*) and p < 0.01 (**).

Journal: Frontiers in Cell and Developmental Biology

Article Title: Integrin α2 is an early marker for osteoclast differentiation that contributes to key steps in osteoclastogenesis

doi: 10.3389/fcell.2024.1448725

Figure Lengend Snippet: Integrin α2β1 deficiency cannot be compensated by OSCAR and results in reduced osteoclastic syncytia formation and hydroxyapatite resorption. (A) qRT-PCR of OSCAR at different time points of differentiation of ER-Hoxb8 ITGA2wt and -ITGA2-KO cells on collagen-I-coated or uncoated glass coverslips (n = 6 and 8, respectively). (B) Quantification of cell fusion after 7 days of differentiation via the number of Hoechst 33342-stained nuclei per cell. More than 7,500 cells in 3 independent experiments were analyzed. A Pearson Chi-squared test revealed a significant difference between the ITGA2wt and ITGA2-KO cell populations ( p < 0.0001), but no significant difference for each of the 2 cell variants between collagen-I and uncoated glass. (C) qRT-PCR of DC-STAMP at different time points of differentiation of Hoxb8-ITGA2wt and -ITGA2-KO cells on collagen-I-coated or uncoated glass coverslips. (D) Resorption areas (light grey) of ER-Hoxb8-derived osteoclasts after 10 days of differentiation on hydroxyapatite-coated coverslips. Cells were removed and remaining hydroxyapatite was silver-stained. Representative images of 8 independent experiments are shown. (E) Biometric quantification of resorption areas in (D) using the trainable WEKA segmentation of Fiji (ImageJ 1.53t), n = 8 ± S.E.M., p < 0.05. (F) qRT-PCR of TRAP at different time points of differentiation of ITGA2wt and ITGA2-KO ER-Hoxb8 cells on collagen-I-coated or uncoated glass coverslips. (G) qRT-PCR of CTSK at different time points of differentiation of ITGA2wt and -ITGA2-KO cells on collagen-I-coated or uncoated glass coverslips. qRT-PCR data (panels C , F , and G ) are represented as mean ± S.E.M. from three to four independent experiments in duplicate with Student’s t-test showing pairwise significances as p < 0.05 (*) and p < 0.01 (**).

Techniques: Quantitative RT-PCR, Staining, Derivative Assay

Expression of osteoclastic marker genes involved in formation of cellular protrusions and syncytia formation. qRT-PCR data of (A) M-Sec that plays a role in TNT formation and fusion, (B) myosin X that is localized on the tips of filopodia, in lamellipodia and podosomes, and (C) the tetraspanin CD9 that contributes to filopodia formation and cell fusion during osteoclastogenesis. Data are mean ± S.E.M. from 3 independent experiments in duplicate with Student’s t-test showing pairwise significances as p < 0.05 (*) and p < 0.01 (**). (D) CD9 deficiency in ITGA2-KO osteoclasts at day 6 of differentiation on collagen-I shown by confocal laser scanning microscopy at 20× magnification. F-actin (red), integrin α2 (green), CD9 (grey), nuclei (blue). Representative images from one out of 4 experiments are shown.

Journal: Frontiers in Cell and Developmental Biology

Article Title: Integrin α2 is an early marker for osteoclast differentiation that contributes to key steps in osteoclastogenesis

doi: 10.3389/fcell.2024.1448725

Figure Lengend Snippet: Expression of osteoclastic marker genes involved in formation of cellular protrusions and syncytia formation. qRT-PCR data of (A) M-Sec that plays a role in TNT formation and fusion, (B) myosin X that is localized on the tips of filopodia, in lamellipodia and podosomes, and (C) the tetraspanin CD9 that contributes to filopodia formation and cell fusion during osteoclastogenesis. Data are mean ± S.E.M. from 3 independent experiments in duplicate with Student’s t-test showing pairwise significances as p < 0.05 (*) and p < 0.01 (**). (D) CD9 deficiency in ITGA2-KO osteoclasts at day 6 of differentiation on collagen-I shown by confocal laser scanning microscopy at 20× magnification. F-actin (red), integrin α2 (green), CD9 (grey), nuclei (blue). Representative images from one out of 4 experiments are shown.

Techniques: Expressing, Marker, Quantitative RT-PCR, Confocal Laser Scanning Microscopy

CD9 only partially colocalizes with integrin α2β1 and its heterogeneous expression on OC precursor determines intercellular contact formation and cell fusion in osteoclastogenesis. (A) Representative image of an ITGA2 expressing non-polarized, rounded OC at day 6 of differentiation on collagen-I showing marked colocalization of integrin α2, CD9 and F-actin in protrusions. (B) Orthogonal view of cropped region marked in (A) with three-fold stretched z -axis to better discern the colocalization of integrin α2 and CD9. (C) Reduced co-localization of integrin α2 and CD9 in filopodia of a polarized (likely motile) OC. (D) Orthogonal view of cropped region marked in (C) with three-fold stretched z -axis to better discern the localization of integrin α2 and CD9 in different planes, with integrin α2 closer to the substrate and CD9 more distal from the substrate. Representative confocal laser scanning microscope image acquired at 40× magnification with integrin α2 (green), CD9 (purple), F-actin (red), nuclei (blue). (E) The ratio of bicellular fusion of heterogeneously CD9 expressing cells to homogeneously CD9-positive cell pairs was determined in two ways. Theoretically, the fusion ratio was calculated using the determined frequency of mononuclear CD9-expressing ITGA2wt cells (4,235 cell from 66 images, bin size: at least 30 cells) as a fully stochastic two-step Bernoulli process. Experimentally, binuclear cell fusion events from ITGA2wt cells grown on collagen-I for 4 days were quantified after immunostaining for CD9 expression and the fusion ratio of heterogeneously CD9-expressing cells to homogenously CD9-positive cells was calculated (>300 binuclear cell fusions on 66 images, with a bin size of at least 9 binuclear syncytia). With a significance level of p = 0.05, the experimentally determined binuclear fusion ratio did not differ significantly from the theoretically calculated one, suggesting that binuclear fusion of differentiating ER-Hoxb8 cells is a stochastic process, independently of cellular CD9 abundance. (F) Intercellular contact sites of fusing ER-Hoxb8 cells may differ with respect to the presence of CD9. After 4 days of differentiation on collagen-I, OCs were immunofluorescently stained for CD9 (green), F-actin (red), nuclei (blue) (left panels). The enlarged section on the right shows a subcellular contact site of two fusing cells (top), which are heterotypic with regard to CD9 expression (bottom, single channel). A representative confocal laser scanning microscope image acquired at 20× magnification from n = 4 independent experiments from a total of more than 40 arbitrarily selected contacting cells is shown.

Journal: Frontiers in Cell and Developmental Biology

Article Title: Integrin α2 is an early marker for osteoclast differentiation that contributes to key steps in osteoclastogenesis

doi: 10.3389/fcell.2024.1448725

Figure Lengend Snippet: CD9 only partially colocalizes with integrin α2β1 and its heterogeneous expression on OC precursor determines intercellular contact formation and cell fusion in osteoclastogenesis. (A) Representative image of an ITGA2 expressing non-polarized, rounded OC at day 6 of differentiation on collagen-I showing marked colocalization of integrin α2, CD9 and F-actin in protrusions. (B) Orthogonal view of cropped region marked in (A) with three-fold stretched z -axis to better discern the colocalization of integrin α2 and CD9. (C) Reduced co-localization of integrin α2 and CD9 in filopodia of a polarized (likely motile) OC. (D) Orthogonal view of cropped region marked in (C) with three-fold stretched z -axis to better discern the localization of integrin α2 and CD9 in different planes, with integrin α2 closer to the substrate and CD9 more distal from the substrate. Representative confocal laser scanning microscope image acquired at 40× magnification with integrin α2 (green), CD9 (purple), F-actin (red), nuclei (blue). (E) The ratio of bicellular fusion of heterogeneously CD9 expressing cells to homogeneously CD9-positive cell pairs was determined in two ways. Theoretically, the fusion ratio was calculated using the determined frequency of mononuclear CD9-expressing ITGA2wt cells (4,235 cell from 66 images, bin size: at least 30 cells) as a fully stochastic two-step Bernoulli process. Experimentally, binuclear cell fusion events from ITGA2wt cells grown on collagen-I for 4 days were quantified after immunostaining for CD9 expression and the fusion ratio of heterogeneously CD9-expressing cells to homogenously CD9-positive cells was calculated (>300 binuclear cell fusions on 66 images, with a bin size of at least 9 binuclear syncytia). With a significance level of p = 0.05, the experimentally determined binuclear fusion ratio did not differ significantly from the theoretically calculated one, suggesting that binuclear fusion of differentiating ER-Hoxb8 cells is a stochastic process, independently of cellular CD9 abundance. (F) Intercellular contact sites of fusing ER-Hoxb8 cells may differ with respect to the presence of CD9. After 4 days of differentiation on collagen-I, OCs were immunofluorescently stained for CD9 (green), F-actin (red), nuclei (blue) (left panels). The enlarged section on the right shows a subcellular contact site of two fusing cells (top), which are heterotypic with regard to CD9 expression (bottom, single channel). A representative confocal laser scanning microscope image acquired at 20× magnification from n = 4 independent experiments from a total of more than 40 arbitrarily selected contacting cells is shown.

Techniques: Expressing, Laser-Scanning Microscopy, Immunostaining, Staining

Journal: International Journal of Molecular Sciences

Article Title: αvβ3 Integrin as a Link between the Development of Fibrosis and Thyroid Hormones in Systemic Sclerosis

doi: 10.3390/ijms24108927

Figure Lengend Snippet: List of antibodies.

Article Snippet: Integrin αv , Rabbit , Polyclonal Ab , Cell Signaling Technology (Danvers, MA, USA) , #4711 , 1:1000 , BSA 5%.

Techniques: Hybridization

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