phospho-stat1 (tyr701) (58d6) rabbit mab  (Cell Signaling Technology Inc)

Journal: Animal Bioscience

Article Title: Transcriptomic analysis identifies CXCL12 as a novel candidate gene for litter size in rabbits

doi: 10.5713/ab.24.0640

Figure Lengend Snippet: CXCL12 and its receptor CXCR4 were expressed and localized in the ovary, with their binding activating the JAK/STAT signaling pathway. (A) Immunofluorescence staining of ovarian follicles revealed that CXCL12 was co-localized with its receptor CXCR4 in ovarian follicles. (B) CXCL12 overexpression significantly promoted the expression of CXCR4 , JAK2 and STAT1 . (C) Knocking down CXCL12 significantly inhibited the expression of CXCR4 , JAK2 and STAT1 . (D) CXCL12 overexpression in GCs promoted the protein expression of CXCR4, JAK2 and STAT1 as well as the phosphorylation of JAK2 and STAT1. When CXCL12 was knocked down, the results were reversed. (E) MSX-122 inhibitor-based treatment further confirmed that CXCL12 promoted the total protein and phosphorylation levels of JAK2 and STAT1 (The t test was used for the above analyses comparing two individual samples. * p<0.05; ** p<0.01; *** p<0.001).

Article Snippet: Protein detection was achieved using the following antibodies: anti-CCND1 mouse monoclonal antibody (1:250, Proteintech), anti-PCNA rabbit polyclonal antibody (1:250, Proteintech), anti-Bcl2 rabbit polyclonal antibody (1:250, Proteintech), anti-Bax rabbit polyclonal antibody (1:250, Proteintech), anti-CITED1 rabbit polyclonal antibody (1:50, Proteintech), anti-WNT10B mouse monoclonal polyclonal antibody (1:250, Proteintech), anti-CXCR4 mouse monoclonal polyclonal antibody (1:250, Proteintech), anti-phospho-JAK2 rabbit monoclonal polyclonal antibody (1:250, Abcam, Cambridge, UK), anti-JAK2 rabbit monoclonal polyclonal antibody (1:250, Abcam), anti-phospho-STAT1 rabbit polyclonal antibody (1:250, Proteintech), anti-STAT1 rabbit polyclonal antibody (1:250, Proteintech), anti-GAPDH mouse monoclonal antibody (1:2,500, Proteintech), 1:1,000 goat anti-rabbit secondary antibody IgG (Proteintech) and 1:1,000 goat anti-mouse secondary antibody IgG (Proteintech).

Techniques: Binding Assay, Immunofluorescence, Staining, Over Expression, Expressing, Phospho-proteomics

Journal: Journal of Neuroinflammation

Article Title: VSIG2 as a novel immunosuppressive ligand interacts with Nectin-2 to regulate T cell responses

doi: 10.1186/s12974-025-03645-7

Figure Lengend Snippet: Anti-Nectin-2 Ab blocks the inhibitory effect of hVSIG2-Ig on T cells. CD3⁺ T cells were isolated by magnetic bead sorting and pre-incubated with 5 µg/ml anti-Nectin-2 antibody. Cells were then plated in 96-well plates pre-coated with anti-CD3/CD28 (1 µg/ml/0.5 µg/ml) and 6400 ng/ml hVSIG2-Ig, followed by culture for 24–72 h. a Schematic illustration of Nectin-2 receptor blockade on T cells using anti-Nectin-2 antibody. b Representative flow cytometry plots of CD69, CD25, and CD40L expression in CD4⁺ and CD8⁺ T cells, with ( c ) quantitative analysis of T cell activation ( n = 3 independent experiments). d Representative flow cytometry plots of Ki67 expression in CD4⁺ and CD8⁺ T cells, with e quantitative analysis of T cell proliferation ( n = 3 independent experiments). f Cytokine microsphere array (CBA) analysis of TNF-α, IFN-γ, IL-17 A, IL-2, and IL-6 in culture supernatants ( n = 3 independent experiments). g Immunoblot analysis of p-STAT1, GBP2, and IRF1 protein expression in T cells, with ( h ) densitometric quantification ( n = 3 independent experiments). Data are presented as mean ± SD. Statistical significance was assessed by one-way ANOVA with Tukey’s test. Source data are provided as a Source Data file

Article Snippet: The membranes were incubated with corresponding primary antibodies: anti‐VSIG2 (1:500; OTI2D8, Thermo Fisher Scientific), anti‐Phospho-STAT1 (Ser727) (1:500; YA149, Medchemexpress), anti‐STAT1 (1:500; YA059, Medchemexpress), anti‐GBP2 (1:500; 11854-1-AP, Proteintech), anti‐GBP5 (1:500; 13220-1-AP, Proteintech), anti‐IRF1 (1:500; ET1602-28, Huabio), anti‐NF‐κB p65 (1:500; HA721307, Huabio), anti‐CD69(1:500; HA500073 , Huabio), anti‐Pro-caspase-1 (1:500; AF5418, Affinity), anti‐NLRP3 (1:500; BF8029, Affinity), anti‐Cleaved-Caspase 1 (Asp296) (1:500; AF4005, Affinity) and anti‐GAPDH (1:500; 60004-1-Ig, Proteintech) overnight at 4 °C.

Techniques: Isolation, Incubation, Flow Cytometry, Expressing, Activation Assay, Western Blot

Journal: Journal of Neuroinflammation

Article Title: VSIG2 as a novel immunosuppressive ligand interacts with Nectin-2 to regulate T cell responses

doi: 10.1186/s12974-025-03645-7

Figure Lengend Snippet: Knockdown of Nectin-2 blocks the inhibitory effect of hVSIG2-Ig on Jurkat cells. a Schematic illustration of hVSIG2-Ig blockade in Jurkat cells with lentiviral-mediated Nectin-2 knockdown ( n = 3 independent experiments). b Flow cytometry analysis of Nectin-2 surface expression in resting and activated Jurkat cells after lentiviral transfection ( n = 3 independent experiments). c Western blot validation of Nectin-2 knockdown efficiency in Jurkat cells ( n = 3 independent experiments). d Representative flow cytometry plots of CD69, CD25, and IL-2 expression in Jurkat cells, with quantitative analysis of T cell activation markers ( n = 3 independent experiments). e Representative flow cytometry plots of Ki67 expression in Jurkat cells, with statistical analysis of proliferation rates ( n = 3 independent experiments). f Western blot analysis of p-STAT1, GBP2, and IRF1 protein levels in Jurkat cells, with g densitometric quantification of band intensities ( n = 3 independent experiments). Data are presented as mean ± SD. Statistical significance was assessed by two-way ANOVA with Tukey’s test

Article Snippet: The membranes were incubated with corresponding primary antibodies: anti‐VSIG2 (1:500; OTI2D8, Thermo Fisher Scientific), anti‐Phospho-STAT1 (Ser727) (1:500; YA149, Medchemexpress), anti‐STAT1 (1:500; YA059, Medchemexpress), anti‐GBP2 (1:500; 11854-1-AP, Proteintech), anti‐GBP5 (1:500; 13220-1-AP, Proteintech), anti‐IRF1 (1:500; ET1602-28, Huabio), anti‐NF‐κB p65 (1:500; HA721307, Huabio), anti‐CD69(1:500; HA500073 , Huabio), anti‐Pro-caspase-1 (1:500; AF5418, Affinity), anti‐NLRP3 (1:500; BF8029, Affinity), anti‐Cleaved-Caspase 1 (Asp296) (1:500; AF4005, Affinity) and anti‐GAPDH (1:500; 60004-1-Ig, Proteintech) overnight at 4 °C.

Techniques: Knockdown, Flow Cytometry, Expressing, Transfection, Western Blot, Biomarker Discovery, Activation Assay

Journal: Journal of Neuroinflammation

Article Title: VSIG2 as a novel immunosuppressive ligand interacts with Nectin-2 to regulate T cell responses

doi: 10.1186/s12974-025-03645-7

Figure Lengend Snippet: Role of GBP2 in VSIG2-mediated T cell activation. Purified CD4 + T cells were isolated from splenocytes of C57BL/6 mice using magnetic bead sorting. Cells were then plated on anti-CD3 antibody (1 µg/ml) and anti-CD28 (0.5 µg/ml) antibodies pre-coated plates and incubated for 24 h in the presence of hVSIG2-Ig (6400 ng/ml) or an equimolar concentration of control Ig. After incubation, the cells were harvested for RNA-Seq and DIA proteomic sequencing ( n = 3 independent experiments). a CD4⁺ T cells were isolated from C57BL/6 mouse spleens by magnetic bead sorting, transduced with lentiviral vectors for GBP2 knockdown or overexpression, and cultured for 48 h. Cells were then transferred to 96-well plates pre-coated with anti-CD3/CD28 antibodies and hVSIG2-Ig for an additional 24 h. b Immunoblot analysis of GBP2 knockdown efficiency in CD4⁺ T cells ( n = 3 independent experiments). c Representative flow cytometry analysis of CD69 expression in CD4⁺ T cells, with statistical quantification of T cell activation ( n = 3 independent experiments). d Immunoblot analysis of p-STAT1, IRF1, and CD69 protein levels in GBP2 knockdown CD4⁺ T cells, with ( e ) semiquantitative analysis ( n = 3 independent experiments). f Immunoblot analysis of GBP2 overexpression in CD4⁺ T cells ( n = 3 independent experiments). g Representative flow cytometry analysis of CD69 expression in CD4⁺ T cells, with statistical quantification of T cell activation ( n = 3 independent experiments). h Immunoblot analysis of p-STAT1, IRF1, and CD69 protein levels in GBP2 overexpressing CD4⁺ T cells, with ( i ) densitometric quantification ( n = 3 independent experiments). Data are presented as mean ± SD. Statistical significance was assessed by two-way ANOVA with Tukey’s test

Article Snippet: The membranes were incubated with corresponding primary antibodies: anti‐VSIG2 (1:500; OTI2D8, Thermo Fisher Scientific), anti‐Phospho-STAT1 (Ser727) (1:500; YA149, Medchemexpress), anti‐STAT1 (1:500; YA059, Medchemexpress), anti‐GBP2 (1:500; 11854-1-AP, Proteintech), anti‐GBP5 (1:500; 13220-1-AP, Proteintech), anti‐IRF1 (1:500; ET1602-28, Huabio), anti‐NF‐κB p65 (1:500; HA721307, Huabio), anti‐CD69(1:500; HA500073 , Huabio), anti‐Pro-caspase-1 (1:500; AF5418, Affinity), anti‐NLRP3 (1:500; BF8029, Affinity), anti‐Cleaved-Caspase 1 (Asp296) (1:500; AF4005, Affinity) and anti‐GAPDH (1:500; 60004-1-Ig, Proteintech) overnight at 4 °C.

Techniques: Activation Assay, Purification, Isolation, Incubation, Concentration Assay, Control, RNA Sequencing, Sequencing, Transduction, Knockdown, Over Expression, Cell Culture, Western Blot, Flow Cytometry, Expressing

Journal: Journal of Neuroinflammation

Article Title: VSIG2 as a novel immunosuppressive ligand interacts with Nectin-2 to regulate T cell responses

doi: 10.1186/s12974-025-03645-7

Figure Lengend Snippet: Anti-Nectin-2 Ab blocks the inhibitory effect of hVSIG2-Ig on T cells. CD3⁺ T cells were isolated by magnetic bead sorting and pre-incubated with 5 µg/ml anti-Nectin-2 antibody. Cells were then plated in 96-well plates pre-coated with anti-CD3/CD28 (1 µg/ml/0.5 µg/ml) and 6400 ng/ml hVSIG2-Ig, followed by culture for 24–72 h. a Schematic illustration of Nectin-2 receptor blockade on T cells using anti-Nectin-2 antibody. b Representative flow cytometry plots of CD69, CD25, and CD40L expression in CD4⁺ and CD8⁺ T cells, with ( c ) quantitative analysis of T cell activation ( n = 3 independent experiments). d Representative flow cytometry plots of Ki67 expression in CD4⁺ and CD8⁺ T cells, with e quantitative analysis of T cell proliferation ( n = 3 independent experiments). f Cytokine microsphere array (CBA) analysis of TNF-α, IFN-γ, IL-17 A, IL-2, and IL-6 in culture supernatants ( n = 3 independent experiments). g Immunoblot analysis of p-STAT1, GBP2, and IRF1 protein expression in T cells, with ( h ) densitometric quantification ( n = 3 independent experiments). Data are presented as mean ± SD. Statistical significance was assessed by one-way ANOVA with Tukey’s test. Source data are provided as a Source Data file

Article Snippet: The membranes were incubated with corresponding primary antibodies: anti‐VSIG2 (1:500; OTI2D8, Thermo Fisher Scientific), anti‐Phospho-STAT1 (Ser727) (1:500; YA149, Medchemexpress), anti‐STAT1 (1:500; YA059, Medchemexpress), anti‐GBP2 (1:500; 11854-1-AP, Proteintech), anti‐GBP5 (1:500; 13220-1-AP, Proteintech), anti‐IRF1 (1:500; ET1602-28, Huabio), anti‐NF‐κB p65 (1:500; HA721307, Huabio), anti‐CD69(1:500; HA500073 , Huabio), anti‐Pro-caspase-1 (1:500; AF5418, Affinity), anti‐NLRP3 (1:500; BF8029, Affinity), anti‐Cleaved-Caspase 1 (Asp296) (1:500; AF4005, Affinity) and anti‐GAPDH (1:500; 60004-1-Ig, Proteintech) overnight at 4 °C.

Techniques: Isolation, Incubation, Flow Cytometry, Expressing, Activation Assay, Western Blot

Journal: Journal of Neuroinflammation

Article Title: VSIG2 as a novel immunosuppressive ligand interacts with Nectin-2 to regulate T cell responses

doi: 10.1186/s12974-025-03645-7

Figure Lengend Snippet: Knockdown of Nectin-2 blocks the inhibitory effect of hVSIG2-Ig on Jurkat cells. a Schematic illustration of hVSIG2-Ig blockade in Jurkat cells with lentiviral-mediated Nectin-2 knockdown ( n = 3 independent experiments). b Flow cytometry analysis of Nectin-2 surface expression in resting and activated Jurkat cells after lentiviral transfection ( n = 3 independent experiments). c Western blot validation of Nectin-2 knockdown efficiency in Jurkat cells ( n = 3 independent experiments). d Representative flow cytometry plots of CD69, CD25, and IL-2 expression in Jurkat cells, with quantitative analysis of T cell activation markers ( n = 3 independent experiments). e Representative flow cytometry plots of Ki67 expression in Jurkat cells, with statistical analysis of proliferation rates ( n = 3 independent experiments). f Western blot analysis of p-STAT1, GBP2, and IRF1 protein levels in Jurkat cells, with g densitometric quantification of band intensities ( n = 3 independent experiments). Data are presented as mean ± SD. Statistical significance was assessed by two-way ANOVA with Tukey’s test

Article Snippet: The membranes were incubated with corresponding primary antibodies: anti‐VSIG2 (1:500; OTI2D8, Thermo Fisher Scientific), anti‐Phospho-STAT1 (Ser727) (1:500; YA149, Medchemexpress), anti‐STAT1 (1:500; YA059, Medchemexpress), anti‐GBP2 (1:500; 11854-1-AP, Proteintech), anti‐GBP5 (1:500; 13220-1-AP, Proteintech), anti‐IRF1 (1:500; ET1602-28, Huabio), anti‐NF‐κB p65 (1:500; HA721307, Huabio), anti‐CD69(1:500; HA500073 , Huabio), anti‐Pro-caspase-1 (1:500; AF5418, Affinity), anti‐NLRP3 (1:500; BF8029, Affinity), anti‐Cleaved-Caspase 1 (Asp296) (1:500; AF4005, Affinity) and anti‐GAPDH (1:500; 60004-1-Ig, Proteintech) overnight at 4 °C.

Techniques: Knockdown, Flow Cytometry, Expressing, Transfection, Western Blot, Biomarker Discovery, Activation Assay

Journal: Journal of Neuroinflammation

Article Title: VSIG2 as a novel immunosuppressive ligand interacts with Nectin-2 to regulate T cell responses

doi: 10.1186/s12974-025-03645-7

Figure Lengend Snippet: Role of GBP2 in VSIG2-mediated T cell activation. Purified CD4 + T cells were isolated from splenocytes of C57BL/6 mice using magnetic bead sorting. Cells were then plated on anti-CD3 antibody (1 µg/ml) and anti-CD28 (0.5 µg/ml) antibodies pre-coated plates and incubated for 24 h in the presence of hVSIG2-Ig (6400 ng/ml) or an equimolar concentration of control Ig. After incubation, the cells were harvested for RNA-Seq and DIA proteomic sequencing ( n = 3 independent experiments). a CD4⁺ T cells were isolated from C57BL/6 mouse spleens by magnetic bead sorting, transduced with lentiviral vectors for GBP2 knockdown or overexpression, and cultured for 48 h. Cells were then transferred to 96-well plates pre-coated with anti-CD3/CD28 antibodies and hVSIG2-Ig for an additional 24 h. b Immunoblot analysis of GBP2 knockdown efficiency in CD4⁺ T cells ( n = 3 independent experiments). c Representative flow cytometry analysis of CD69 expression in CD4⁺ T cells, with statistical quantification of T cell activation ( n = 3 independent experiments). d Immunoblot analysis of p-STAT1, IRF1, and CD69 protein levels in GBP2 knockdown CD4⁺ T cells, with ( e ) semiquantitative analysis ( n = 3 independent experiments). f Immunoblot analysis of GBP2 overexpression in CD4⁺ T cells ( n = 3 independent experiments). g Representative flow cytometry analysis of CD69 expression in CD4⁺ T cells, with statistical quantification of T cell activation ( n = 3 independent experiments). h Immunoblot analysis of p-STAT1, IRF1, and CD69 protein levels in GBP2 overexpressing CD4⁺ T cells, with ( i ) densitometric quantification ( n = 3 independent experiments). Data are presented as mean ± SD. Statistical significance was assessed by two-way ANOVA with Tukey’s test

Article Snippet: The membranes were incubated with corresponding primary antibodies: anti‐VSIG2 (1:500; OTI2D8, Thermo Fisher Scientific), anti‐Phospho-STAT1 (Ser727) (1:500; YA149, Medchemexpress), anti‐STAT1 (1:500; YA059, Medchemexpress), anti‐GBP2 (1:500; 11854-1-AP, Proteintech), anti‐GBP5 (1:500; 13220-1-AP, Proteintech), anti‐IRF1 (1:500; ET1602-28, Huabio), anti‐NF‐κB p65 (1:500; HA721307, Huabio), anti‐CD69(1:500; HA500073 , Huabio), anti‐Pro-caspase-1 (1:500; AF5418, Affinity), anti‐NLRP3 (1:500; BF8029, Affinity), anti‐Cleaved-Caspase 1 (Asp296) (1:500; AF4005, Affinity) and anti‐GAPDH (1:500; 60004-1-Ig, Proteintech) overnight at 4 °C.

Techniques: Activation Assay, Purification, Isolation, Incubation, Concentration Assay, Control, RNA Sequencing, Sequencing, Transduction, Knockdown, Over Expression, Cell Culture, Western Blot, Flow Cytometry, Expressing

Journal: Life Science Alliance

Article Title: STAT1-IFITM3 promotes autophagy in epithelial cells to control Cryptosporidium parvum infection

doi: 10.26508/lsa.202503200

Figure Lengend Snippet: (A) An inhibitor of STAT1, Fludarabine (10 μM), was used in uninfected and C. parvum -infected HCT-8 cells. Western blotting analysis of STAT1, p-STAT1, IFITM3, and LC3B levels in uninfected, STAT1 inhibited, infected, and STAT1 inhibited infected groups. (B, C, D, E) Histogram analysis of STAT1 (B), p-STAT1 (C), IFITM3 (D), and LC3B (E) in the four groups. (F, G) qRT-PCR analysis of STAT1 and IFITM3 expression levels in the four groups. (H, I) Parasite numbers and cell numbers were quantified by qRT-PCR assays at 24 hpi. (J) Western blotting analysis of STAT1, p-STAT1 and IFITM3 levels in the control, siIFITM3, infected, and siIFITM3 infected groups. (K, L, M) Histogram analysis of IFITM3 (K), STAT1 (L), and p-STAT1 (M) in the four groups. (N) Coimmunoprecipitation to determine whether IFITM3 directly interacted with STAT1. At least three independent experiments were carried out. Data are presented as means ± SD, and differences were identified using ANOVA (* P < 0.05, ** P < 0.01, *** P < 0.001, ns, no significant difference).

Article Snippet: Then, the PVDF membranes were incubated overnight at 4°C with primary antibodies recognizing IFITM3 (Proteintech), LC3B (Abcam), p62 (Abcam), ATG5 (Abcam), ATG7 (Abcam), β-actin (ACTB) (Proteintech), STAT1 (Proteintech), Phospho(p)-STAT1 (Tyr701) (Proteintech), IL-8 (Abcam), and TNF-α (Proteintech).

Techniques: Infection, Western Blot, Quantitative RT-PCR, Expressing, Control

Journal: iScience

Article Title: AF6 regulates intestinal IgA via crosstalk between intestinal epithelial cells and immune cells in inflammatory bowel disease

doi: 10.1016/j.isci.2025.112658

Figure Lengend Snippet: AF6 regulates the expression of MHC II by modulating the expression of STAT1 in intestinal epithelial cells (IECs) (A) The protein levels of components of the IFN-γ-related signaling pathway were detected by immunoblotting of colon tissues from Af6 f/f and Af6 ΔIEC mice maintained on 2.5% DSS for 7 days. (B) qPCR was used to assess STAT1 transcript levels in colon tissues from Af6 f/f and Af6 ΔIEC mice maintained on 2.5% DSS for 7 days. (C and D) (C) The mRNA levels of STAT1 and its downstream target genes (as assessed by qPCR) and (D) the protein levels of STAT1 and proteins encoded by its downstream target genes (as assessed by immunoblotting) were determined in organoids generated from colon tissues of Af6 f/f and Af6 ΔIEC mice, as measured before and after IFN-γ treatment. (E) Co-immunoprecipitation (co-IP) was used to detect endogenous interactions between AF6 and IRF1 in IECs. (F) 293T cells were co-transfected with constructs encoding hemagglutinin-tagged AF6 (HA-AF6) and Flag peptide-tagged IRF1 (Flag-IRF1); their interaction domains were detected by co-IP. (G) Immunoblotting was used to assess the expression of IRF1 and proteins encoded by its downstream genes in HT29 cells with or without IFN-γ exposure. (H) HT29 cells were transfected with constructs encoding AF6 with no or 3 nuclear localization signaling (NLS) domains (ΔNLS-AF6 and 3×NLS-AF6, respectively), and the expression and localization of IRF1 and proteins encoded by its downstream genes were assessed by immunoblotting following the separation of the nuclear and cytoplasmic fractions. Data are expressed as mean ± SEM. Pairwise comparisons between groups were conducted using two-tailed non-paired Student’s t tests. p -values were shown in the panel, and p < 0.05 indicates a significant difference.

Article Snippet: Phospho-Stat1 (Tyr701) (58D6) Rabbit mAb , Cell Signaling Technology , Cat#9167.

Techniques: Expressing, Western Blot, Generated, Immunoprecipitation, Co-Immunoprecipitation Assay, Transfection, Construct, Two Tailed Test