integrin subunit beta 2  (Proteintech)

Journal: Thoracic Cancer

Article Title: Epithelial‐Mesenchymal Plasticity in the D‐Meso‐Sonobe Mesothelioma Cell Line: A Putative Model of Epithelial–Mesenchymal Transition in Mesothelioma

doi: 10.1111/1759-7714.70091

Figure Lengend Snippet: Immunohistochemical staining of xenoplanted D‐Meso‐Sonobe tumor cells. Zeb1 immunoreactivity was barely found in xenoplanted tumor cells (a and b). Furthermore, nuclear Zeb1 immunoreactivity was found in various tumor cells at the cancer invasion front (indicated by ☆ in a and b). Cytoplasmic yes‐associated protein (YAP) immunoreactivity was found in center of the xenoplanted tumor (c), whereas nuclear YAP immunoreactivity was also found in tumor invasion front (d). Integrin Subunit Alpha V and Actin alpha 2 immunoreactivities were focally found in the xenoplanted tumor (e and f, respectively). LOXL1 immunoreactivity was found at the invasion front near muscle cells (indicated by white arrowhead), while minimum staining was observed in the non‐invasion front (indicated by black arrow). Scale bars represent 100 μm (a and g) and 50 μm (b–f).

Article Snippet: The rabbit antibodies against Integrin Subunit Alpha V (ITGAV) (cat. no. 27096‐1‐AP), and actin alpha 2 (ACTA2) (cat. no. 14395‐1‐AP) were purchased from Proteintech.

Techniques: Immunohistochemical staining, Staining

Journal: Frontiers in Immunology

Article Title: TSP-1-CD47-integrin α4β1 axis drives T cell infiltration and synovial inflammation in rheumatoid arthritis

doi: 10.3389/fimmu.2025.1524304

Figure Lengend Snippet: Expression of CD47, its two ligands and several integrin subunits in synovial tissue and peripheral blood samples of human RA patients. (A–J) Violin Plots for increased expression of CD47, SIRP-α, TSP-1, integrin subunits (α4, αM, αv, αL, β1, β2, β3) in synovial tissues from RA patients compared with healthy people. Data were based on RA patient synovial tissue samples (RA=230, Normal=59) published on GPL96, GPL570, GPL11154, GPL1708, GPL10558, and GPL91 platforms from NCBI-GEO. (K–O) . Violin Plots for increased expression of CD47, SIRP-α, TSP-1, and integrin subunits (α4 and αL) in peripheral blood samples from RA patients compared with healthy people. Data were based on RA patient peripheral blood samples (RA=1238, Normal=120) published on GPL6947, GPL570, GPL20171, and GPL13158 platforms from NCBI-GEO.

Article Snippet: Cell lysate was incubated with an anti-CD47 antibody (SANTA CRUZ, SC-12730), an anti-integrin subunit α4 antibody (Novus, NBP2-50445) or an anti-integrin subunit β1 antibody (Abcom, ab24693) that have been properly diluted according to the instructions of the products.

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: TSP-1-CD47-integrin α4β1 axis drives T cell infiltration and synovial inflammation in rheumatoid arthritis

doi: 10.3389/fimmu.2025.1524304

Figure Lengend Snippet: Expression of CD47, its two ligands and several integrin subunits in rat synovial tissues. On day 28 of the animal experiment shown in Figure 2 , rats were sacrificed and synovial tissues of the hind paws were taken for western-blot, quantitative real-time PCR, and RNAseq analysis. (A) Photo of western-blot for the expression of CD47, SIRP-α, TSP-1, integrin subunit α4, αM, αv, αL, β1, β2 and β3. (B–K) Expression of CD47 (B) , SIRP-α (C) , TSP-1 (D) , integrin subunit α4 (E) , αM (F) , αv (G) , αL (H) , β1 (I) , β2 (J) and β3 (K) on an mRNA level in synovial membranes of rats in the four groups were shown and compared. For (B–K) , n=3 (three replicates for detection of each molecule). All of the data show mean ± SD. **P < 0.01. (L) Differential expression of CD47, its two ligands (SIRP-α and TSP-1), and several integrin subunits in synovial membranes of rats in the four groups by RNAseq analysis (n=3, samples of three animals for each group). Gene expression heatmap was generated by Cluster 3.0 with the hierarchical method and R 4.0.3 with the heatmap method.

Article Snippet: Cell lysate was incubated with an anti-CD47 antibody (SANTA CRUZ, SC-12730), an anti-integrin subunit α4 antibody (Novus, NBP2-50445) or an anti-integrin subunit β1 antibody (Abcom, ab24693) that have been properly diluted according to the instructions of the products.

Techniques: Expressing, Western Blot, Real-time Polymerase Chain Reaction, Quantitative Proteomics, Gene Expression, Generated

Journal: Frontiers in Immunology

Article Title: TSP-1-CD47-integrin α4β1 axis drives T cell infiltration and synovial inflammation in rheumatoid arthritis

doi: 10.3389/fimmu.2025.1524304

Figure Lengend Snippet: Peptide 4N1K engagement on CD47 increased integrin α4β1 activation and function on human Jurkat cells. (A) CD47, α4 integrin, and cell nucleus were probed and stained by an APC conjugated anti-CD47 antibody, a FITC conjugated anti-α4 antibody or Hoechst staining and their merge signal confirmed the interaction between CD47 and α4 integrin on the cell surface (400×). (B–D) . Western-blot analysis of the bands for CD47, α4, and β1 integrin subunit after immunoprecipitation with an anti-CD47 (B) , anti-α4 (C) , or anti-β1 (D) antibody. Cell lysate which was negatively labeled on the left was precipitated with an isotype control antibody. The whole-cell lysate on the right was named input. (E) Peptide 4N1K treatment increased the adhesion of human Jurkat cells to immobilized VCAM-1 (n=3, three replicate samples for each experimental condition). Cells in the control sample were not incubated with peptides. Typical photos of adhesion cells under various treatment conditions (×100) were shown on the left. (F) Peptide 4N1K treatment increased the migration of human Jurkat cells to immobilized VCAM-1 with SDF-1 as a chemoattractant (n=3, three replicate samples for each experimental condition). Typical photos of migrated cells under various treatment conditions (×200) were shown on the left. (G–J) Peptide 4N1K increased the percent of “extended form” integrin α4β1 with detection by an anti-active form α4β1 antibody. Cells were treated with peptide 4KGG (I) , 4N1K (J) , or without peptide treatment (H) and then were probed with an anti-active form integrin α4β1 antibody and thereafter a FITC-labeled secondary antibody. Human Jurkat cells incubated with an isotype antibody were used as an isotype control sample (G) . Statistical analysis of up-regulation of integrin α4β1 active form was shown in (K) (n=3, three detections for the same experimental condition). Data show mean ± SEM. **P < 0.01. Total expression of integrin α4β1 on Jurkat cells was confirmed with the use of a FITC conjugated rabbit anti-human integrin α4β1 monoclonal antibody (M) . Jurkat cells incubated with an isotype antibody were used as an isotype control sample (L) .

Article Snippet: Cell lysate was incubated with an anti-CD47 antibody (SANTA CRUZ, SC-12730), an anti-integrin subunit α4 antibody (Novus, NBP2-50445) or an anti-integrin subunit β1 antibody (Abcom, ab24693) that have been properly diluted according to the instructions of the products.

Techniques: Activation Assay, Staining, Western Blot, Immunoprecipitation, Labeling, Control, Incubation, Migration, Expressing

Journal: Frontiers in Immunology

Article Title: TSP-1-CD47-integrin α4β1 axis drives T cell infiltration and synovial inflammation in rheumatoid arthritis

doi: 10.3389/fimmu.2025.1524304

Figure Lengend Snippet: Inside-out signaling pathways lead to integrin α4β1 activation. A monoclonal antibody α-PSα4 was used for the detection of the Ser988-phosphorylated form of the α4 cytoplasmic domain. (A) Peptide 4N1K and TSP increased the amount of phosphorylated α4 subunit in Jurkat cells whereas peptide 4KGG had no effect. (B) PKAI and PP2 inhibited 4N1K effect on α4 subunit phosphorylation in Jurkat cells whereas PTX had no effect. Peptide 4N1K and 4KGG treatment did not change the amount of intracellular cAMP (C) or IP3 (D) in human Jurkat cells (n=3, three replicate samples for each experimental condition). Results are presented as the mean ± SD. For CD3+ T cells isolated from wild-type rats, peptide 4N1K and TSP increased the amount of phosphorylated α4 subunit whereas peptide 4KGG had no effect (E) . PKAI and PP2 inhibited 4N1K effect on α4 subunit phosphorylation whereas PTX had no effect (F) . The amount of intracellular cAMP (H) or IP3 (I) was not significantly changed in wild-type rat CD3+ T cells under treatment with peptide 4N1K, 4KGG, or without peptide treatment (n=3, three replicate samples for each experimental condition). Results are presented as the mean ± SD. For CD3+ T cells isolated from Cd47 knockout rats, the amount of phosphorylated α4 subunit was undetectable whether the cells were treated with peptide 4N1K, TSP (J) , or 4N1K in combination with PKAI, PTX, or PP2 (K) . The total amount of integrin α4β1 was not changed under peptide 4N1K or its combination with PKAI, PTX, or PP2 treatment for CD3+T cells isolated from wild-type rats (G) and Cd47 knockout rats (L) .

Article Snippet: Cell lysate was incubated with an anti-CD47 antibody (SANTA CRUZ, SC-12730), an anti-integrin subunit α4 antibody (Novus, NBP2-50445) or an anti-integrin subunit β1 antibody (Abcom, ab24693) that have been properly diluted according to the instructions of the products.

Techniques: Protein-Protein interactions, Activation Assay, Phospho-proteomics, Isolation, Knock-Out

Journal: Frontiers in Immunology

Article Title: TSP-1-CD47-integrin α4β1 axis drives T cell infiltration and synovial inflammation in rheumatoid arthritis

doi: 10.3389/fimmu.2025.1524304

Figure Lengend Snippet: The therapeutic effect of local injection of CD47 antibody, PKA inhibitor, G protein inhibitor, or Src kinase inhibitor on arthritis development. Six groups of wild-type rats were included (n=5) and for panels (A–D) the solid black line stands for the normal rat group, the solid orange line for the model group, the dashed red line for the CD47 antibody treatment group, the solid blue line for the PKA inhibitor (H89) treatment group, the dashed purple line for the G protein inhibitor treatment group and the green line for the Src kinase inhibitor (PP2) treatment group. (A–D) . Swelling of hind left paws (A) and right paws (B) , arthritis score (C) and clinic score (D) of rats during the experiment were shown. On day 28 of the animal experiment, rats were sacrificed and synovial tissues of the hind left paws were taken and used as samples for western-blot analysis. Photos of western-blot for CD47, SIRP-α, TSP-1, integrin subunit α4, αM, αv, αL, β1, β2, and β3 under PKA inhibitor H89, G protein inhibitor PTX, and Src kinase inhibitor PP2 treatment (E) and CD47 antibody treatment (F) were shown. Photos of western-blot for markers of T cells (CD3), neutrophils (Ly6G), macrophages (CD68), and B cells (CD45RA) in synovial membranes of rats under PKA inhibitor H89, G protein inhibitor PTX, and Src kinase inhibitor PP2 treatment (G) and CD47 antibody treatment (H) were shown.

Article Snippet: Cell lysate was incubated with an anti-CD47 antibody (SANTA CRUZ, SC-12730), an anti-integrin subunit α4 antibody (Novus, NBP2-50445) or an anti-integrin subunit β1 antibody (Abcom, ab24693) that have been properly diluted according to the instructions of the products.

Techniques: Injection, Western Blot

Journal: Frontiers in Immunology

Article Title: TSP-1-CD47-integrin α4β1 axis drives T cell infiltration and synovial inflammation in rheumatoid arthritis

doi: 10.3389/fimmu.2025.1524304

Figure Lengend Snippet: “TSP-1-CD47-integrin α4β1” trimolecular co-action model to explain the important role of CD47 in RA. CD47 expressed on the surface of T cells interacts with TSP-1 scattered on the blood vessel to promote integrin α4β1 (on T cell surface) interaction with VCAM-1 present on the surface of vascular endothelial cells, thereby promoting T cells infiltration into the joint synovial tissue and accelerating arthritis development.

Article Snippet: Cell lysate was incubated with an anti-CD47 antibody (SANTA CRUZ, SC-12730), an anti-integrin subunit α4 antibody (Novus, NBP2-50445) or an anti-integrin subunit β1 antibody (Abcom, ab24693) that have been properly diluted according to the instructions of the products.

Techniques:

Journal: Frontiers in Immunology

Article Title: TSP-1-CD47-integrin α4β1 axis drives T cell infiltration and synovial inflammation in rheumatoid arthritis

doi: 10.3389/fimmu.2025.1524304

Figure Lengend Snippet: FIGURE 1 Expression of CD47, its two ligands and several integrin subunits in synovial tissue and peripheral blood samples of human RA patients. (A–J) Violin Plots for increased expression of CD47, SIRP-a, TSP-1, integrin subunits (a4, aM, av, aL, b1, b2, b3) in synovial tissues from RA patients compared with healthy people. Data were based on RA patient synovial tissue samples (RA=230, Normal=59) published on GPL96, GPL570, GPL11154, GPL1708, GPL10558, and GPL91 platforms from NCBI-GEO. (K–O). Violin Plots for increased expression of CD47, SIRP-a, TSP-1, and integrin subunits (a4 and aL) in peripheral blood samples from RA patients compared with healthy people. Data were based on RA patient peripheral blood samples (RA=1238, Normal=120) published on GPL6947, GPL570, GPL20171, and GPL13158 platforms from NCBI-GEO.

Article Snippet: Monoclonal antibodies specific for integrin subunit a4 (sc-376334), b2 (sc-8420), p-a4 (sc23943), and IL-23 (sc-271279) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: TSP-1-CD47-integrin α4β1 axis drives T cell infiltration and synovial inflammation in rheumatoid arthritis

doi: 10.3389/fimmu.2025.1524304

Figure Lengend Snippet: FIGURE 3 Expression of CD47, its two ligands and several integrin subunits in rat synovial tissues. On day 28 of the animal experiment shown in Figure 2, rats were sacrificed and synovial tissues of the hind paws were taken for western-blot, quantitative real-time PCR, and RNAseq analysis. (A) Photo of western-blot for the expression of CD47, SIRP-a, TSP-1, integrin subunit a4, aM, av, aL, b1, b2 and b3. (B–K) Expression of CD47 (B), SIRP-a (C), TSP-1 (D), integrin subunit a4 (E), aM (F), av (G), aL (H), b1 (I), b2 (J) and b3 (K) on an mRNA level in synovial membranes of rats in the four groups were shown and compared. For (B–K), n=3 (three replicates for detection of each molecule). All of the data show mean ± SD. **P < 0.01. (L) Differential expression of CD47, its two ligands (SIRP-a and TSP-1), and several integrin subunits in synovial membranes of rats in the four groups by RNAseq analysis (n=3, samples of three animals for each group). Gene expression heatmap was generated by Cluster 3.0 with the hierarchical method and R 4.0.3 with the heatmap method.

Article Snippet: Monoclonal antibodies specific for integrin subunit a4 (sc-376334), b2 (sc-8420), p-a4 (sc23943), and IL-23 (sc-271279) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Expressing, Western Blot, Real-time Polymerase Chain Reaction, Quantitative Proteomics, Gene Expression, Generated

Journal: Frontiers in Immunology

Article Title: TSP-1-CD47-integrin α4β1 axis drives T cell infiltration and synovial inflammation in rheumatoid arthritis

doi: 10.3389/fimmu.2025.1524304

Figure Lengend Snippet: FIGURE 5 Peptide 4N1K engagement on CD47 increased integrin a4b1 activation and function on human Jurkat cells. (A) CD47, a4 integrin, and cell nucleus were probed and stained by an APC conjugated anti-CD47 antibody, a FITC conjugated anti-a4 antibody or Hoechst staining and their merge signal confirmed the interaction between CD47 and a4 integrin on the cell surface (400×). (B–D). Western-blot analysis of the bands for CD47, a4, and b1 integrin subunit after immunoprecipitation with an anti-CD47 (B), anti-a4 (C), or anti-b1 (D) antibody. Cell lysate which was negatively labeled on the left was precipitated with an isotype control antibody. The whole-cell lysate on the right was named input. (E) Peptide 4N1K treatment increased the adhesion of human Jurkat cells to immobilized VCAM-1 (n=3, three replicate samples for each experimental condition). Cells in the control sample were not incubated with peptides. Typical photos of adhesion cells under various treatment conditions (×100) were shown on the left. (F) Peptide 4N1K treatment increased the migration of human Jurkat cells to immobilized VCAM-1 with SDF-1 as a chemoattractant (n=3, three replicate samples for each experimental condition). Typical photos of migrated cells under various treatment conditions (×200) were shown on the left. (G–J) Peptide 4N1K increased the percent of “extended form” integrin a4b1 with detection by an anti-active form a4b1 antibody. Cells were treated with peptide 4KGG (I), 4N1K (J), or without peptide treatment (H) and then were probed with an anti-active form integrin a4b1 antibody and thereafter a FITC-labeled secondary antibody. Human Jurkat cells incubated with an isotype antibody were used as an isotype control sample (G). Statistical analysis of up-regulation of integrin a4b1 active form was shown in (K) (n=3, three detections for the same experimental condition). Data show mean ± SEM. **P < 0.01. Total expression of integrin a4b1 on Jurkat cells was confirmed with the use of a FITC conjugated rabbit anti-human integrin a4b1 monoclonal antibody (M). Jurkat cells incubated with an isotype antibody were used as an isotype control sample (L).

Article Snippet: Monoclonal antibodies specific for integrin subunit a4 (sc-376334), b2 (sc-8420), p-a4 (sc23943), and IL-23 (sc-271279) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Activation Assay, Staining, Western Blot, Immunoprecipitation, Labeling, Control, Incubation, Migration, Expressing

Journal: Frontiers in Immunology

Article Title: TSP-1-CD47-integrin α4β1 axis drives T cell infiltration and synovial inflammation in rheumatoid arthritis

doi: 10.3389/fimmu.2025.1524304

Figure Lengend Snippet: FIGURE 6 Inside-out signaling pathways lead to integrin a4b1 activation. A monoclonal antibody a-PSa4 was used for the detection of the Ser988- phosphorylated form of the a4 cytoplasmic domain. (A) Peptide 4N1K and TSP increased the amount of phosphorylated a4 subunit in Jurkat cells whereas peptide 4KGG had no effect. (B) PKAI and PP2 inhibited 4N1K effect on a4 subunit phosphorylation in Jurkat cells whereas PTX had no effect. Peptide 4N1K and 4KGG treatment did not change the amount of intracellular cAMP (C) or IP3 (D) in human Jurkat cells (n=3, three replicate samples for each experimental condition). Results are presented as the mean ± SD. For CD3+ T cells isolated from wild-type rats, peptide 4N1K and TSP increased the amount of phosphorylated a4 subunit whereas peptide 4KGG had no effect (E). PKAI and PP2 inhibited 4N1K effect on a4 subunit phosphorylation whereas PTX had no effect (F). The amount of intracellular cAMP (H) or IP3 (I) was not significantly changed in wild-type rat CD3+ T cells under treatment with peptide 4N1K, 4KGG, or without peptide treatment (n=3, three replicate samples for each experimental condition). Results are presented as the mean ± SD. For CD3+ T cells isolated from Cd47 knockout rats, the amount of phosphorylated a4 subunit was undetectable whether the cells were treated with peptide 4N1K, TSP (J), or 4N1K in combination with PKAI, PTX, or PP2 (K). The total amount of integrin a4b1 was not changed under peptide 4N1K or its combination with PKAI, PTX, or PP2 treatment for CD3+T cells isolated from wild-type rats (G) and Cd47 knockout rats (L).

Article Snippet: Monoclonal antibodies specific for integrin subunit a4 (sc-376334), b2 (sc-8420), p-a4 (sc23943), and IL-23 (sc-271279) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Protein-Protein interactions, Activation Assay, Phospho-proteomics, Isolation, Knock-Out

Journal: Frontiers in Immunology

Article Title: TSP-1-CD47-integrin α4β1 axis drives T cell infiltration and synovial inflammation in rheumatoid arthritis

doi: 10.3389/fimmu.2025.1524304

Figure Lengend Snippet: FIGURE 7 The therapeutic effect of local injection of CD47 antibody, PKA inhibitor, G protein inhibitor, or Src kinase inhibitor on arthritis development. Six groups of wild-type rats were included (n=5) and for panels (A–D) the solid black line stands for the normal rat group, the solid orange line for the model group, the dashed red line for the CD47 antibody treatment group, the solid blue line for the PKA inhibitor (H89) treatment group, the dashed purple line for the G protein inhibitor treatment group and the green line for the Src kinase inhibitor (PP2) treatment group. (A–D). Swelling of hind left paws (A) and right paws (B), arthritis score (C) and clinic score (D) of rats during the experiment were shown. On day 28 of the animal experiment, rats were sacrificed and synovial tissues of the hind left paws were taken and used as samples for western-blot analysis. Photos of western-blot for CD47, SIRP-a, TSP-1, integrin subunit a4, aM, av, aL, b1, b2, and b3 under PKA inhibitor H89, G protein inhibitor PTX, and Src kinase inhibitor PP2 treatment (E) and CD47 antibody treatment (F) were shown. Photos of western-blot for markers of T cells (CD3), neutrophils (Ly6G), macrophages (CD68), and B cells (CD45RA) in synovial membranes of rats under PKA inhibitor H89, G protein inhibitor PTX, and Src kinase inhibitor PP2 treatment (G) and CD47 antibody treatment (H) were shown.

Article Snippet: Monoclonal antibodies specific for integrin subunit a4 (sc-376334), b2 (sc-8420), p-a4 (sc23943), and IL-23 (sc-271279) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Injection, Western Blot

Journal: Frontiers in Immunology

Article Title: TSP-1-CD47-integrin α4β1 axis drives T cell infiltration and synovial inflammation in rheumatoid arthritis

doi: 10.3389/fimmu.2025.1524304

Figure Lengend Snippet: FIGURE 8 “TSP-1-CD47-integrin a4b1” trimolecular co-action model to explain the important role of CD47 in RA. CD47 expressed on the surface of T cells interacts with TSP-1 scattered on the blood vessel to promote integrin a4b1 (on T cell surface) interaction with VCAM-1 present on the surface of vascular endothelial cells, thereby promoting T cells infiltration into the joint synovial tissue and accelerating arthritis development.

Article Snippet: Monoclonal antibodies specific for integrin subunit a4 (sc-376334), b2 (sc-8420), p-a4 (sc23943), and IL-23 (sc-271279) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques:

Journal: Frontiers in Immunology

Article Title: TSP-1-CD47-integrin α4β1 axis drives T cell infiltration and synovial inflammation in rheumatoid arthritis

doi: 10.3389/fimmu.2025.1524304

Figure Lengend Snippet: Expression of CD47, its two ligands and several integrin subunits in synovial tissue and peripheral blood samples of human RA patients. (A–J) Violin Plots for increased expression of CD47, SIRP-α, TSP-1, integrin subunits (α4, αM, αv, αL, β1, β2, β3) in synovial tissues from RA patients compared with healthy people. Data were based on RA patient synovial tissue samples (RA=230, Normal=59) published on GPL96, GPL570, GPL11154, GPL1708, GPL10558, and GPL91 platforms from NCBI-GEO. (K–O) . Violin Plots for increased expression of CD47, SIRP-α, TSP-1, and integrin subunits (α4 and αL) in peripheral blood samples from RA patients compared with healthy people. Data were based on RA patient peripheral blood samples (RA=1238, Normal=120) published on GPL6947, GPL570, GPL20171, and GPL13158 platforms from NCBI-GEO.

Article Snippet: Monoclonal antibodies specific for integrin subunit α4 (sc-376334), β2 (sc-8420), p-α4 (sc-23943), and IL-23 (sc-271279) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: TSP-1-CD47-integrin α4β1 axis drives T cell infiltration and synovial inflammation in rheumatoid arthritis

doi: 10.3389/fimmu.2025.1524304

Figure Lengend Snippet: Expression of CD47, its two ligands and several integrin subunits in rat synovial tissues. On day 28 of the animal experiment shown in Figure 2 , rats were sacrificed and synovial tissues of the hind paws were taken for western-blot, quantitative real-time PCR, and RNAseq analysis. (A) Photo of western-blot for the expression of CD47, SIRP-α, TSP-1, integrin subunit α4, αM, αv, αL, β1, β2 and β3. (B–K) Expression of CD47 (B) , SIRP-α (C) , TSP-1 (D) , integrin subunit α4 (E) , αM (F) , αv (G) , αL (H) , β1 (I) , β2 (J) and β3 (K) on an mRNA level in synovial membranes of rats in the four groups were shown and compared. For (B–K) , n=3 (three replicates for detection of each molecule). All of the data show mean ± SD. **P < 0.01. (L) Differential expression of CD47, its two ligands (SIRP-α and TSP-1), and several integrin subunits in synovial membranes of rats in the four groups by RNAseq analysis (n=3, samples of three animals for each group). Gene expression heatmap was generated by Cluster 3.0 with the hierarchical method and R 4.0.3 with the heatmap method.

Article Snippet: Monoclonal antibodies specific for integrin subunit α4 (sc-376334), β2 (sc-8420), p-α4 (sc-23943), and IL-23 (sc-271279) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Expressing, Western Blot, Real-time Polymerase Chain Reaction, Quantitative Proteomics, Gene Expression, Generated

Journal: Frontiers in Immunology

Article Title: TSP-1-CD47-integrin α4β1 axis drives T cell infiltration and synovial inflammation in rheumatoid arthritis

doi: 10.3389/fimmu.2025.1524304

Figure Lengend Snippet: Peptide 4N1K engagement on CD47 increased integrin α4β1 activation and function on human Jurkat cells. (A) CD47, α4 integrin, and cell nucleus were probed and stained by an APC conjugated anti-CD47 antibody, a FITC conjugated anti-α4 antibody or Hoechst staining and their merge signal confirmed the interaction between CD47 and α4 integrin on the cell surface (400×). (B–D) . Western-blot analysis of the bands for CD47, α4, and β1 integrin subunit after immunoprecipitation with an anti-CD47 (B) , anti-α4 (C) , or anti-β1 (D) antibody. Cell lysate which was negatively labeled on the left was precipitated with an isotype control antibody. The whole-cell lysate on the right was named input. (E) Peptide 4N1K treatment increased the adhesion of human Jurkat cells to immobilized VCAM-1 (n=3, three replicate samples for each experimental condition). Cells in the control sample were not incubated with peptides. Typical photos of adhesion cells under various treatment conditions (×100) were shown on the left. (F) Peptide 4N1K treatment increased the migration of human Jurkat cells to immobilized VCAM-1 with SDF-1 as a chemoattractant (n=3, three replicate samples for each experimental condition). Typical photos of migrated cells under various treatment conditions (×200) were shown on the left. (G–J) Peptide 4N1K increased the percent of “extended form” integrin α4β1 with detection by an anti-active form α4β1 antibody. Cells were treated with peptide 4KGG (I) , 4N1K (J) , or without peptide treatment (H) and then were probed with an anti-active form integrin α4β1 antibody and thereafter a FITC-labeled secondary antibody. Human Jurkat cells incubated with an isotype antibody were used as an isotype control sample (G) . Statistical analysis of up-regulation of integrin α4β1 active form was shown in (K) (n=3, three detections for the same experimental condition). Data show mean ± SEM. **P < 0.01. Total expression of integrin α4β1 on Jurkat cells was confirmed with the use of a FITC conjugated rabbit anti-human integrin α4β1 monoclonal antibody (M) . Jurkat cells incubated with an isotype antibody were used as an isotype control sample (L) .

Article Snippet: Monoclonal antibodies specific for integrin subunit α4 (sc-376334), β2 (sc-8420), p-α4 (sc-23943), and IL-23 (sc-271279) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Activation Assay, Staining, Western Blot, Immunoprecipitation, Labeling, Control, Incubation, Migration, Expressing

Journal: Frontiers in Immunology

Article Title: TSP-1-CD47-integrin α4β1 axis drives T cell infiltration and synovial inflammation in rheumatoid arthritis

doi: 10.3389/fimmu.2025.1524304

Figure Lengend Snippet: Inside-out signaling pathways lead to integrin α4β1 activation. A monoclonal antibody α-PSα4 was used for the detection of the Ser988-phosphorylated form of the α4 cytoplasmic domain. (A) Peptide 4N1K and TSP increased the amount of phosphorylated α4 subunit in Jurkat cells whereas peptide 4KGG had no effect. (B) PKAI and PP2 inhibited 4N1K effect on α4 subunit phosphorylation in Jurkat cells whereas PTX had no effect. Peptide 4N1K and 4KGG treatment did not change the amount of intracellular cAMP (C) or IP3 (D) in human Jurkat cells (n=3, three replicate samples for each experimental condition). Results are presented as the mean ± SD. For CD3+ T cells isolated from wild-type rats, peptide 4N1K and TSP increased the amount of phosphorylated α4 subunit whereas peptide 4KGG had no effect (E) . PKAI and PP2 inhibited 4N1K effect on α4 subunit phosphorylation whereas PTX had no effect (F) . The amount of intracellular cAMP (H) or IP3 (I) was not significantly changed in wild-type rat CD3+ T cells under treatment with peptide 4N1K, 4KGG, or without peptide treatment (n=3, three replicate samples for each experimental condition). Results are presented as the mean ± SD. For CD3+ T cells isolated from Cd47 knockout rats, the amount of phosphorylated α4 subunit was undetectable whether the cells were treated with peptide 4N1K, TSP (J) , or 4N1K in combination with PKAI, PTX, or PP2 (K) . The total amount of integrin α4β1 was not changed under peptide 4N1K or its combination with PKAI, PTX, or PP2 treatment for CD3+T cells isolated from wild-type rats (G) and Cd47 knockout rats (L) .

Article Snippet: Monoclonal antibodies specific for integrin subunit α4 (sc-376334), β2 (sc-8420), p-α4 (sc-23943), and IL-23 (sc-271279) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Protein-Protein interactions, Activation Assay, Phospho-proteomics, Isolation, Knock-Out

Journal: Frontiers in Immunology

Article Title: TSP-1-CD47-integrin α4β1 axis drives T cell infiltration and synovial inflammation in rheumatoid arthritis

doi: 10.3389/fimmu.2025.1524304

Figure Lengend Snippet: The therapeutic effect of local injection of CD47 antibody, PKA inhibitor, G protein inhibitor, or Src kinase inhibitor on arthritis development. Six groups of wild-type rats were included (n=5) and for panels (A–D) the solid black line stands for the normal rat group, the solid orange line for the model group, the dashed red line for the CD47 antibody treatment group, the solid blue line for the PKA inhibitor (H89) treatment group, the dashed purple line for the G protein inhibitor treatment group and the green line for the Src kinase inhibitor (PP2) treatment group. (A–D) . Swelling of hind left paws (A) and right paws (B) , arthritis score (C) and clinic score (D) of rats during the experiment were shown. On day 28 of the animal experiment, rats were sacrificed and synovial tissues of the hind left paws were taken and used as samples for western-blot analysis. Photos of western-blot for CD47, SIRP-α, TSP-1, integrin subunit α4, αM, αv, αL, β1, β2, and β3 under PKA inhibitor H89, G protein inhibitor PTX, and Src kinase inhibitor PP2 treatment (E) and CD47 antibody treatment (F) were shown. Photos of western-blot for markers of T cells (CD3), neutrophils (Ly6G), macrophages (CD68), and B cells (CD45RA) in synovial membranes of rats under PKA inhibitor H89, G protein inhibitor PTX, and Src kinase inhibitor PP2 treatment (G) and CD47 antibody treatment (H) were shown.

Article Snippet: Monoclonal antibodies specific for integrin subunit α4 (sc-376334), β2 (sc-8420), p-α4 (sc-23943), and IL-23 (sc-271279) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Injection, Western Blot

Journal: Frontiers in Immunology

Article Title: TSP-1-CD47-integrin α4β1 axis drives T cell infiltration and synovial inflammation in rheumatoid arthritis

doi: 10.3389/fimmu.2025.1524304

Figure Lengend Snippet: “TSP-1-CD47-integrin α4β1” trimolecular co-action model to explain the important role of CD47 in RA. CD47 expressed on the surface of T cells interacts with TSP-1 scattered on the blood vessel to promote integrin α4β1 (on T cell surface) interaction with VCAM-1 present on the surface of vascular endothelial cells, thereby promoting T cells infiltration into the joint synovial tissue and accelerating arthritis development.

Article Snippet: Monoclonal antibodies specific for integrin subunit α4 (sc-376334), β2 (sc-8420), p-α4 (sc-23943), and IL-23 (sc-271279) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques:

Journal: Frontiers in Immunology

Article Title: TSP-1-CD47-integrin α4β1 axis drives T cell infiltration and synovial inflammation in rheumatoid arthritis

doi: 10.3389/fimmu.2025.1524304

Figure Lengend Snippet: Expression of CD47, its two ligands and several integrin subunits in synovial tissue and peripheral blood samples of human RA patients. (A–J) Violin Plots for increased expression of CD47, SIRP-α, TSP-1, integrin subunits (α4, αM, αv, αL, β1, β2, β3) in synovial tissues from RA patients compared with healthy people. Data were based on RA patient synovial tissue samples (RA=230, Normal=59) published on GPL96, GPL570, GPL11154, GPL1708, GPL10558, and GPL91 platforms from NCBI-GEO. (K–O) . Violin Plots for increased expression of CD47, SIRP-α, TSP-1, and integrin subunits (α4 and αL) in peripheral blood samples from RA patients compared with healthy people. Data were based on RA patient peripheral blood samples (RA=1238, Normal=120) published on GPL6947, GPL570, GPL20171, and GPL13158 platforms from NCBI-GEO.

Article Snippet: After blocking of the membrane, total integrin subunit α and phosphorylated subunit α of integrin α4β1 were probed with an anti-integrin subunit α antibody (sc-376334, SANTA CRUZ) or an anti-phosphorylated subunit α antibody (sc-23943, SANTA CRUZ) overnight at 4°C, respectively.

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: TSP-1-CD47-integrin α4β1 axis drives T cell infiltration and synovial inflammation in rheumatoid arthritis

doi: 10.3389/fimmu.2025.1524304

Figure Lengend Snippet: Expression of CD47, its two ligands and several integrin subunits in rat synovial tissues. On day 28 of the animal experiment shown in Figure 2 , rats were sacrificed and synovial tissues of the hind paws were taken for western-blot, quantitative real-time PCR, and RNAseq analysis. (A) Photo of western-blot for the expression of CD47, SIRP-α, TSP-1, integrin subunit α4, αM, αv, αL, β1, β2 and β3. (B–K) Expression of CD47 (B) , SIRP-α (C) , TSP-1 (D) , integrin subunit α4 (E) , αM (F) , αv (G) , αL (H) , β1 (I) , β2 (J) and β3 (K) on an mRNA level in synovial membranes of rats in the four groups were shown and compared. For (B–K) , n=3 (three replicates for detection of each molecule). All of the data show mean ± SD. **P < 0.01. (L) Differential expression of CD47, its two ligands (SIRP-α and TSP-1), and several integrin subunits in synovial membranes of rats in the four groups by RNAseq analysis (n=3, samples of three animals for each group). Gene expression heatmap was generated by Cluster 3.0 with the hierarchical method and R 4.0.3 with the heatmap method.

Article Snippet: After blocking of the membrane, total integrin subunit α and phosphorylated subunit α of integrin α4β1 were probed with an anti-integrin subunit α antibody (sc-376334, SANTA CRUZ) or an anti-phosphorylated subunit α antibody (sc-23943, SANTA CRUZ) overnight at 4°C, respectively.

Techniques: Expressing, Western Blot, Real-time Polymerase Chain Reaction, Quantitative Proteomics, Gene Expression, Generated

Journal: Frontiers in Immunology

Article Title: TSP-1-CD47-integrin α4β1 axis drives T cell infiltration and synovial inflammation in rheumatoid arthritis

doi: 10.3389/fimmu.2025.1524304

Figure Lengend Snippet: Peptide 4N1K engagement on CD47 increased integrin α4β1 activation and function on human Jurkat cells. (A) CD47, α4 integrin, and cell nucleus were probed and stained by an APC conjugated anti-CD47 antibody, a FITC conjugated anti-α4 antibody or Hoechst staining and their merge signal confirmed the interaction between CD47 and α4 integrin on the cell surface (400×). (B–D) . Western-blot analysis of the bands for CD47, α4, and β1 integrin subunit after immunoprecipitation with an anti-CD47 (B) , anti-α4 (C) , or anti-β1 (D) antibody. Cell lysate which was negatively labeled on the left was precipitated with an isotype control antibody. The whole-cell lysate on the right was named input. (E) Peptide 4N1K treatment increased the adhesion of human Jurkat cells to immobilized VCAM-1 (n=3, three replicate samples for each experimental condition). Cells in the control sample were not incubated with peptides. Typical photos of adhesion cells under various treatment conditions (×100) were shown on the left. (F) Peptide 4N1K treatment increased the migration of human Jurkat cells to immobilized VCAM-1 with SDF-1 as a chemoattractant (n=3, three replicate samples for each experimental condition). Typical photos of migrated cells under various treatment conditions (×200) were shown on the left. (G–J) Peptide 4N1K increased the percent of “extended form” integrin α4β1 with detection by an anti-active form α4β1 antibody. Cells were treated with peptide 4KGG (I) , 4N1K (J) , or without peptide treatment (H) and then were probed with an anti-active form integrin α4β1 antibody and thereafter a FITC-labeled secondary antibody. Human Jurkat cells incubated with an isotype antibody were used as an isotype control sample (G) . Statistical analysis of up-regulation of integrin α4β1 active form was shown in (K) (n=3, three detections for the same experimental condition). Data show mean ± SEM. **P < 0.01. Total expression of integrin α4β1 on Jurkat cells was confirmed with the use of a FITC conjugated rabbit anti-human integrin α4β1 monoclonal antibody (M) . Jurkat cells incubated with an isotype antibody were used as an isotype control sample (L) .

Article Snippet: After blocking of the membrane, total integrin subunit α and phosphorylated subunit α of integrin α4β1 were probed with an anti-integrin subunit α antibody (sc-376334, SANTA CRUZ) or an anti-phosphorylated subunit α antibody (sc-23943, SANTA CRUZ) overnight at 4°C, respectively.

Techniques: Activation Assay, Staining, Western Blot, Immunoprecipitation, Labeling, Control, Incubation, Migration, Expressing

Journal: Frontiers in Immunology

Article Title: TSP-1-CD47-integrin α4β1 axis drives T cell infiltration and synovial inflammation in rheumatoid arthritis

doi: 10.3389/fimmu.2025.1524304

Figure Lengend Snippet: Inside-out signaling pathways lead to integrin α4β1 activation. A monoclonal antibody α-PSα4 was used for the detection of the Ser988-phosphorylated form of the α4 cytoplasmic domain. (A) Peptide 4N1K and TSP increased the amount of phosphorylated α4 subunit in Jurkat cells whereas peptide 4KGG had no effect. (B) PKAI and PP2 inhibited 4N1K effect on α4 subunit phosphorylation in Jurkat cells whereas PTX had no effect. Peptide 4N1K and 4KGG treatment did not change the amount of intracellular cAMP (C) or IP3 (D) in human Jurkat cells (n=3, three replicate samples for each experimental condition). Results are presented as the mean ± SD. For CD3+ T cells isolated from wild-type rats, peptide 4N1K and TSP increased the amount of phosphorylated α4 subunit whereas peptide 4KGG had no effect (E) . PKAI and PP2 inhibited 4N1K effect on α4 subunit phosphorylation whereas PTX had no effect (F) . The amount of intracellular cAMP (H) or IP3 (I) was not significantly changed in wild-type rat CD3+ T cells under treatment with peptide 4N1K, 4KGG, or without peptide treatment (n=3, three replicate samples for each experimental condition). Results are presented as the mean ± SD. For CD3+ T cells isolated from Cd47 knockout rats, the amount of phosphorylated α4 subunit was undetectable whether the cells were treated with peptide 4N1K, TSP (J) , or 4N1K in combination with PKAI, PTX, or PP2 (K) . The total amount of integrin α4β1 was not changed under peptide 4N1K or its combination with PKAI, PTX, or PP2 treatment for CD3+T cells isolated from wild-type rats (G) and Cd47 knockout rats (L) .

Article Snippet: After blocking of the membrane, total integrin subunit α and phosphorylated subunit α of integrin α4β1 were probed with an anti-integrin subunit α antibody (sc-376334, SANTA CRUZ) or an anti-phosphorylated subunit α antibody (sc-23943, SANTA CRUZ) overnight at 4°C, respectively.

Techniques: Protein-Protein interactions, Activation Assay, Phospho-proteomics, Isolation, Knock-Out

Journal: Frontiers in Immunology

Article Title: TSP-1-CD47-integrin α4β1 axis drives T cell infiltration and synovial inflammation in rheumatoid arthritis

doi: 10.3389/fimmu.2025.1524304

Figure Lengend Snippet: The therapeutic effect of local injection of CD47 antibody, PKA inhibitor, G protein inhibitor, or Src kinase inhibitor on arthritis development. Six groups of wild-type rats were included (n=5) and for panels (A–D) the solid black line stands for the normal rat group, the solid orange line for the model group, the dashed red line for the CD47 antibody treatment group, the solid blue line for the PKA inhibitor (H89) treatment group, the dashed purple line for the G protein inhibitor treatment group and the green line for the Src kinase inhibitor (PP2) treatment group. (A–D) . Swelling of hind left paws (A) and right paws (B) , arthritis score (C) and clinic score (D) of rats during the experiment were shown. On day 28 of the animal experiment, rats were sacrificed and synovial tissues of the hind left paws were taken and used as samples for western-blot analysis. Photos of western-blot for CD47, SIRP-α, TSP-1, integrin subunit α4, αM, αv, αL, β1, β2, and β3 under PKA inhibitor H89, G protein inhibitor PTX, and Src kinase inhibitor PP2 treatment (E) and CD47 antibody treatment (F) were shown. Photos of western-blot for markers of T cells (CD3), neutrophils (Ly6G), macrophages (CD68), and B cells (CD45RA) in synovial membranes of rats under PKA inhibitor H89, G protein inhibitor PTX, and Src kinase inhibitor PP2 treatment (G) and CD47 antibody treatment (H) were shown.

Article Snippet: After blocking of the membrane, total integrin subunit α and phosphorylated subunit α of integrin α4β1 were probed with an anti-integrin subunit α antibody (sc-376334, SANTA CRUZ) or an anti-phosphorylated subunit α antibody (sc-23943, SANTA CRUZ) overnight at 4°C, respectively.

Techniques: Injection, Western Blot

Journal: Frontiers in Immunology

Article Title: TSP-1-CD47-integrin α4β1 axis drives T cell infiltration and synovial inflammation in rheumatoid arthritis

doi: 10.3389/fimmu.2025.1524304

Figure Lengend Snippet: “TSP-1-CD47-integrin α4β1” trimolecular co-action model to explain the important role of CD47 in RA. CD47 expressed on the surface of T cells interacts with TSP-1 scattered on the blood vessel to promote integrin α4β1 (on T cell surface) interaction with VCAM-1 present on the surface of vascular endothelial cells, thereby promoting T cells infiltration into the joint synovial tissue and accelerating arthritis development.

Article Snippet: After blocking of the membrane, total integrin subunit α and phosphorylated subunit α of integrin α4β1 were probed with an anti-integrin subunit α antibody (sc-376334, SANTA CRUZ) or an anti-phosphorylated subunit α antibody (sc-23943, SANTA CRUZ) overnight at 4°C, respectively.

Techniques: