α4β1  (R&D Systems)

Journal: Neurotherapeutics

Article Title: Cellular fibronectin exacerbates α-synuclein aggregation via integrin alpha4beta1 mediated PARP1 and SCD elevation

doi: 10.1016/j.neurot.2025.e00729

Figure Lengend Snippet: cFn induced PARP1 activation via EIIIA-integrin α4β1 interaction, which subsequently contributed to pathological α-syn aggregation and mitochondrial dysfunction in SH-SY5Y cells. (a) Representative western blots and quantification of nNOS and PARP1 in SH-SY5Y cells coated with cFn and treated with different receptor inhibitors (five independent replicates). (b) Quantification of NO release in SH-SY5Y cells treated with cFn or different receptor inhibitors (five independent replicates). (c) Representative western blots and quantification of the TX-insoluble α-syn oligomer and Ser129-phosphorylated α-syn in SH-SY5Y cells (five independent replicates). (d–e) Representative images and quantification of PARP1 (d) and γ-H2AX (e) staining in SH-SY5Y cells 12 ​h after L-NAME or PBS pretreatment (five independent replicates). DAPI (blue) was used for nuclear staining. Scale bar: 100 ​μm. (f) Representative western blots and quantification of PAR and PARP1 in SH-SY5Y cells coated with cFn or AF38Pep at different concentration (five independent replicates). (g) Representative coimmunoprecipitation images showing an interaction between Fn and the integrin α4 subunit in SH-SY5Y cells, and the quantification of Fn and α4 subunit level in the coimmunoprecipitation samples was performed (five independent replicates). All data are presented as the mean ​± ​s.e.m. Differences among 2 groups were analysed by Unpaired Student's t -test, while differences among multiple groups were analysed by ANOVA followed by Tukey's post hoc test. ∗P ​< ​0.05, ∗∗P ​< ​0.01, ∗∗∗P ​< ​0.0005, ∗∗∗∗P ​< ​0.0001, and ns, not significant.

Article Snippet: To prevent interactions between Fn and different receptors, SH-SY5Y cells were pretreated with the integrin α5β1 receptor inhibitor ATN-161 trifluoroacetate salt (MCE HY-13535A), the integrin α4β1 receptor inhibitor TR-14035 (MCE HY-15770), or the TLR4 inhibitor TLR4-IN-C34 (MCE HY-107575) following the manufacturers' protocols.

Techniques: Activation Assay, Western Blot, Staining, Concentration Assay

Journal: Neurotherapeutics

Article Title: Cellular fibronectin exacerbates α-synuclein aggregation via integrin alpha4beta1 mediated PARP1 and SCD elevation

doi: 10.1016/j.neurot.2025.e00729

Figure Lengend Snippet: cFn induced FAs and TAGs elevation by increasing SCD, which contributed to pathological α-syn aggregation. LC‒MS/MS Analysis generated data were performed in A-D. (a–b) Changes in FAs in the SNpc of cFn-injected mice compared with those in healthy mice were determined by hierarchical clustering analysis (a) and matchstick analysis (b) (n ​= ​6), (c–d) Changes in TAGs in the SNpc of cFn-injected mice compared with healthy mice were determined by hierarchical clustering analysis (c) and matchstick analysis (d) (n ​= ​6). In the image of hierarchical clustering analysis, red plots represent upregulation, and blue plots represent downregulation. In the matchstick analysis, red lines represent upregulation, while blue lines represent downregulation. ∗P ​< ​0.05, ∗∗P ​< ​0.01. FAs species was described in ; (e) Representative Oil Red O staining images and relative quantification of the positive area of lipid droplets in SH-SY5Y cells coated with cFn or treated with an integrin α4β1 inhibitor (five independent replicates). Oli Red O staining was detected in bright field channel. Scale bar: 25 ​μm. (f–g) The levels of TAGs (f) and FAs (g) in SH-SY5Y cells coated with cFn or treated with an integrin α4β1 inhibitor were measured (three independent replicates). (h) Representative immunoblots and quantification of SCD in the SNpc of the control, MPTP, MPTP ​+ ​AAV-Fn1-shRNA, and MPTP ​+ ​AAV-NC groups (n ​= ​5). (i) Representative immunoblots and quantification of SCD in SH-SY5Y cells coated with cFn or treated with an integrin α4β1 inhibitor (five independent replicates). (j) Representative western blots and quantification of SCD in the SNpc of mice injected with cFn or treated with AF38Pep (five independent replicates). (k–l) Representative coimmunoprecipitation images showing an interaction between SCD and the α4 subunit in the SNpc of healthy mice or cFn injected mice, and the quantification of α4 subunit or SCD level in the coimmunoprecipitation samples was performed (n ​= ​5). (m) Representative western blots and quantification of the TX-insoluble α-syn oligomer and Ser129-phosphorylated α-syn in α-syn-overexpressing SH-SY5Y cells coated with cFn or treated with an SCD inhibitor (five independent replicates). All data are presented as the mean ​± ​s.e.m. Differences among 2 groups were analysed by Unpaired Student's t -test, while differences among multiple groups were analysed by ANOVA followed by Tukey's post hoc test. ∗P ​< ​0.05, ∗∗P ​< ​0.01, ∗∗∗P ​< ​0.0005, ∗∗∗∗P ​< ​0.0001, and ns, not significant.

Article Snippet: To prevent interactions between Fn and different receptors, SH-SY5Y cells were pretreated with the integrin α5β1 receptor inhibitor ATN-161 trifluoroacetate salt (MCE HY-13535A), the integrin α4β1 receptor inhibitor TR-14035 (MCE HY-15770), or the TLR4 inhibitor TLR4-IN-C34 (MCE HY-107575) following the manufacturers' protocols.

Techniques: Generated, Injection, Staining, Quantitative Proteomics, Western Blot, Control, shRNA

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: Total expression of integrin α4β1 on wild-type rat CD3+ T cells was confirmed with a flow cytometry method with the use of a rabbit anti-rat integrin α4β1 monoclonal antibody (8440T, Cell Signaling Technology) and Alexa Fluor 488 conjugated Goat Anti-Rabbit IgG(H+L) (FMS-RBaf48801, Fcmacs).

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: Total expression of integrin α4β1 on wild-type rat CD3+ T cells was confirmed with a flow cytometry method with the use of a rabbit anti-rat integrin α4β1 monoclonal antibody (8440T, Cell Signaling Technology) and Alexa Fluor 488 conjugated Goat Anti-Rabbit IgG(H+L) (FMS-RBaf48801, Fcmacs).

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: Total expression of integrin α4β1 on wild-type rat CD3+ T cells was confirmed with a flow cytometry method with the use of a rabbit anti-rat integrin α4β1 monoclonal antibody (8440T, Cell Signaling Technology) and Alexa Fluor 488 conjugated Goat Anti-Rabbit IgG(H+L) (FMS-RBaf48801, Fcmacs).

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: Total expression of integrin α4β1 on wild-type rat CD3+ T cells was confirmed with a flow cytometry method with the use of a rabbit anti-rat integrin α4β1 monoclonal antibody (8440T, Cell Signaling Technology) and Alexa Fluor 488 conjugated Goat Anti-Rabbit IgG(H+L) (FMS-RBaf48801, Fcmacs).

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: Total expression of integrin α4β1 on wild-type rat CD3+ T cells was confirmed with a flow cytometry method with the use of a rabbit anti-rat integrin α4β1 monoclonal antibody (8440T, Cell Signaling Technology) and Alexa Fluor 488 conjugated Goat Anti-Rabbit IgG(H+L) (FMS-RBaf48801, Fcmacs).

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: Total expression of integrin α4β1 on wild-type rat CD3+ T cells was confirmed with a flow cytometry method with the use of a rabbit anti-rat integrin α4β1 monoclonal antibody (8440T, Cell Signaling Technology) and Alexa Fluor 488 conjugated Goat Anti-Rabbit IgG(H+L) (FMS-RBaf48801, Fcmacs).

Techniques: