phospho tie2  (Bioss)

Journal: Cell & Bioscience

Article Title: Tie2-expressing monocytes/macrophages promote angiogenesis in chronically ischaemic brain tissue

doi: 10.1186/s13578-025-01401-1

Figure Lengend Snippet: Results of molecular biology experiments confirming the miR-126-5p/TRPS1/ANGPT2 pathway in vitro. A The predicted miR-126-5p binding sites in the 3'UTR of TRPS1 mRNA, the designed sequences of TRPS1 wt and mut, and the sequences of miR-126-5p from multiple species. B The results of DLR assays were determined by the relative luciferase activities (n = 3, one-way ANOVA and Šídák's multiple comparisons test). C Western blot showing the enrichment of AGO2 in the RIP assay. D Column chart showing the expression levels of miR-126-5p and TRPS1 mRNA in the precipitate determined by qRT‒PCR (n = 3, one-way ANOVA and Šídák's multiple comparisons test). E Agarose gel electrophoresis image showing the molecular size of DNA after sonication (approximately 200–400 bp). F Column chart showing the expression levels of the Angpt2 promoter in the precipitate determined by qRT‒PCR (n = 3, one-way ANOVA and Šídák's multiple comparisons test). G Representative western blot showing the relative expression of TRPS1, ANGPT2, pTie2, and Tie2 in the HUVECs in each group (normalized to β-actin expression). H Densitometric analyses of the relative expression of TRPS1, ANGPT2, and pTie2/Tie2 (n = 3, one-way ANOVA and Šídák's multiple comparisons test). The error bars represent the ± SDs. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. ChIP: chromatin immunoprecipitation; DLR: dual luciferase reporter; RIP: RNA immunoprecipitation

Article Snippet: After being blocked for 2 h with Tris-buffered saline (TBS) containing 5% nonfat skim milk (Beyotime, China) or BSA (Beyotime, China), the membranes were incubated with primary antibodies against TRPS1 (1:1000, 21938-1-AP, Proteintech, China), ANGPT2 (1:1000, 24613-1-AP, Proteintech, China), Tie2 (1:1000, DF7500, Affinity, USA), phospho-Tie2 (1:1000, bs-12262R, Bioss, China), CD11b (1:1000, sc-1186, Santa Cruz, USA), VEGFA (1:1000, 19003-1-AP, Proteintech, China), IGF1 (1:1000, ab106836, Abcam, UK), CD31 (1:1000, sc-20071, Santa Cruz, USA), or β-actin (1:20000, 66009-1-Ig, Proteintech, China) in TBS containing 0.2% Tween-20 (TBST) diluted overnight at 4 °C.

Techniques: In Vitro, Binding Assay, Luciferase, Western Blot, Expressing, Agarose Gel Electrophoresis, Sonication, Chromatin Immunoprecipitation, RNA Immunoprecipitation

Journal: Cell & Bioscience

Article Title: Tie2-expressing monocytes/macrophages promote angiogenesis in chronically ischaemic brain tissue

doi: 10.1186/s13578-025-01401-1

Figure Lengend Snippet: Changes in TEMs from the IJV blood of CICD patients. A Representative DSA and CTP images showing angiogenesis and CBP improvement in CICD patients with Matsushima Grade-A revascularization and Matsushima Grade-C revascularization. B Representative flow cytometric dot plots of TEMs from the IJV blood of patients with intracranial aneurysms (control group), CICD patients with Matsushima Grade-A revascularization and CICD patients with Matsushima Grade-C revascularization. C Scatter plot showing the differences among the three groups (n = 10, one-way ANOVA and Tukey's multiple comparisons test). The error bars represent the ± SDs. CBP: cerebral blood perfusion; CICD: chronically ischaemic cerebrovascular disease; CTP: computed tomography perfusion; DSA: digital subtraction angiography; IJV: internal jugular vein; TEMs: Tie2-expressing monocytes/macrophages

Article Snippet: After being blocked for 2 h with Tris-buffered saline (TBS) containing 5% nonfat skim milk (Beyotime, China) or BSA (Beyotime, China), the membranes were incubated with primary antibodies against TRPS1 (1:1000, 21938-1-AP, Proteintech, China), ANGPT2 (1:1000, 24613-1-AP, Proteintech, China), Tie2 (1:1000, DF7500, Affinity, USA), phospho-Tie2 (1:1000, bs-12262R, Bioss, China), CD11b (1:1000, sc-1186, Santa Cruz, USA), VEGFA (1:1000, 19003-1-AP, Proteintech, China), IGF1 (1:1000, ab106836, Abcam, UK), CD31 (1:1000, sc-20071, Santa Cruz, USA), or β-actin (1:20000, 66009-1-Ig, Proteintech, China) in TBS containing 0.2% Tween-20 (TBST) diluted overnight at 4 °C.

Techniques: Control, Computed Tomography, Expressing

Journal: Cell & Bioscience

Article Title: Tie2-expressing monocytes/macrophages promote angiogenesis in chronically ischaemic brain tissue

doi: 10.1186/s13578-025-01401-1

Figure Lengend Snippet: The results of cell coculture showing the pro-angiogenesis effect of TEMs. A Representative flow cytometric dot plot identifying CD14 + PBMCs, TEMs and TNMs. B Representative immunofluorescence images identifying TEMs (Tie2 + /CD14 + ) and TNMs (Tie2 − /CD14 + ). Bar = 20 μm. C Schematic diagram of the two methods for coculture. D Representative confocal images showing the results of the EdU assay for each group. Bar = 50 μm. E The results were quantified by the percentage of EdU-positive HUVECs per field (n = 3, two-way ANOVA and Šídák's multiple comparisons test). F Representative flow cytometric dot plot showing the results of the apoptosis assay for each group. G Column chart showing the degree of apoptosis in each group (n = 3, two-way ANOVA and Šídák's multiple comparisons test). H Representative fields showing the results of the tube formation assays for each group. Bar = 200 μm. The results were quantified by I the total length, number of branches, and number of nodes (n = 3, one-way ANOVA and Tukey's multiple comparisons test). The error bars represent the ± SDs. EdU: ethynyl deoxyuridine; HUVECs: human umbilical vein endothelial cells; PBMCs: peripheral blood mononuclear cells; TEMs: Tie2-expressing monocytes/macrophages; TNMs: Tie2-negative monocytes/macrophages

Article Snippet: After being blocked for 2 h with Tris-buffered saline (TBS) containing 5% nonfat skim milk (Beyotime, China) or BSA (Beyotime, China), the membranes were incubated with primary antibodies against TRPS1 (1:1000, 21938-1-AP, Proteintech, China), ANGPT2 (1:1000, 24613-1-AP, Proteintech, China), Tie2 (1:1000, DF7500, Affinity, USA), phospho-Tie2 (1:1000, bs-12262R, Bioss, China), CD11b (1:1000, sc-1186, Santa Cruz, USA), VEGFA (1:1000, 19003-1-AP, Proteintech, China), IGF1 (1:1000, ab106836, Abcam, UK), CD31 (1:1000, sc-20071, Santa Cruz, USA), or β-actin (1:20000, 66009-1-Ig, Proteintech, China) in TBS containing 0.2% Tween-20 (TBST) diluted overnight at 4 °C.

Techniques: Immunofluorescence, EdU Assay, Apoptosis Assay, Expressing

Journal: Cell & Bioscience

Article Title: Tie2-expressing monocytes/macrophages promote angiogenesis in chronically ischaemic brain tissue

doi: 10.1186/s13578-025-01401-1

Figure Lengend Snippet: Immunofluorescence and western blotting results showing the expression of relevant cytokines in the CIBT of 2VO + EMS rats. A Representative triple immunofluorescence staining images showing ANGPT2 + , Tie2 + , and CD11b + cells in the CIBT in each group (white arrows indicate TEMs). Bar = 50 µm. B Counts of ANGPT2 + cells in each group (n = 3, one-way ANOVA and Šídák's multiple comparisons test). C Counts of TEMs (Tie2 + and CD11b + ) in each group (n = 9, one-way ANOVA and Šídák's multiple comparisons test). D Representative western blot showing the relative expression of TRPS1, ANGPT2, Tie2, CD11b, VEGFA, IGF1, and CD31 in the CIBT adjacent to the TM tissue in each group (normalized to β-actin expression). E Densitometric analyses of the relative expression of TRPS1, ANGPT2, Tie2, CD11b, VEGFA, IGF1, and CD31 (n = 3, one-way ANOVA and Šídák's multiple comparisons test; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001). The error bars represent the ± SDs. CIBT: chronically ischaemic brain tissue; EMS: encephalomyosynangiosis; TEMs: Tie2-expressing monocytes/macrophages; 2VO: 2-vessel occlusion

Article Snippet: After being blocked for 2 h with Tris-buffered saline (TBS) containing 5% nonfat skim milk (Beyotime, China) or BSA (Beyotime, China), the membranes were incubated with primary antibodies against TRPS1 (1:1000, 21938-1-AP, Proteintech, China), ANGPT2 (1:1000, 24613-1-AP, Proteintech, China), Tie2 (1:1000, DF7500, Affinity, USA), phospho-Tie2 (1:1000, bs-12262R, Bioss, China), CD11b (1:1000, sc-1186, Santa Cruz, USA), VEGFA (1:1000, 19003-1-AP, Proteintech, China), IGF1 (1:1000, ab106836, Abcam, UK), CD31 (1:1000, sc-20071, Santa Cruz, USA), or β-actin (1:20000, 66009-1-Ig, Proteintech, China) in TBS containing 0.2% Tween-20 (TBST) diluted overnight at 4 °C.

Techniques: Immunofluorescence, Western Blot, Expressing, Staining

Journal: Cell & Bioscience

Article Title: Tie2-expressing monocytes/macrophages promote angiogenesis in chronically ischaemic brain tissue

doi: 10.1186/s13578-025-01401-1

Figure Lengend Snippet: Immunofluorescence results showing the expression of pro-angiogenesis factors in the CIBT of 2VO + EMS rats. A Representative triple immunofluorescence staining images showing Tie2 + , CD11b + , and VEGFA + cells and in the CIBT in each group (white arrows indicate TEMs). Bar = 50 µm. B Counts of VEGF + cells in each group (n = 3, one-way ANOVA and Šídák's multiple comparisons test). C Representative triple immunofluorescence staining images showing Tie2 + , CD11b + , and IGF1 + cells in the CIBT in each group (white arrows indicate TEMs). Bar = 50 µm. D Counts of IGF1 + cells in each group (n = 3, one-way ANOVA and Šídák's multiple comparisons test). CIBT: chronically ischaemic brain tissue; EMS: encephalomyosynangiosis; TEMs: Tie2-expressing monocytes/macrophages; 2VO: 2-vessel occlusion

Article Snippet: After being blocked for 2 h with Tris-buffered saline (TBS) containing 5% nonfat skim milk (Beyotime, China) or BSA (Beyotime, China), the membranes were incubated with primary antibodies against TRPS1 (1:1000, 21938-1-AP, Proteintech, China), ANGPT2 (1:1000, 24613-1-AP, Proteintech, China), Tie2 (1:1000, DF7500, Affinity, USA), phospho-Tie2 (1:1000, bs-12262R, Bioss, China), CD11b (1:1000, sc-1186, Santa Cruz, USA), VEGFA (1:1000, 19003-1-AP, Proteintech, China), IGF1 (1:1000, ab106836, Abcam, UK), CD31 (1:1000, sc-20071, Santa Cruz, USA), or β-actin (1:20000, 66009-1-Ig, Proteintech, China) in TBS containing 0.2% Tween-20 (TBST) diluted overnight at 4 °C.

Techniques: Immunofluorescence, Expressing, Staining

Journal: Cell & Bioscience

Article Title: Tie2-expressing monocytes/macrophages promote angiogenesis in chronically ischaemic brain tissue

doi: 10.1186/s13578-025-01401-1

Figure Lengend Snippet: Schematic diagram showing that Tie2-expressing monocytes/macrophages are recruited to chronically ischaemic brain tissue via the miR-126-5p/TRPS1/ANGPT2 pathway to promote the proliferation of endothelial cell and angiogenesis

Article Snippet: After being blocked for 2 h with Tris-buffered saline (TBS) containing 5% nonfat skim milk (Beyotime, China) or BSA (Beyotime, China), the membranes were incubated with primary antibodies against TRPS1 (1:1000, 21938-1-AP, Proteintech, China), ANGPT2 (1:1000, 24613-1-AP, Proteintech, China), Tie2 (1:1000, DF7500, Affinity, USA), phospho-Tie2 (1:1000, bs-12262R, Bioss, China), CD11b (1:1000, sc-1186, Santa Cruz, USA), VEGFA (1:1000, 19003-1-AP, Proteintech, China), IGF1 (1:1000, ab106836, Abcam, UK), CD31 (1:1000, sc-20071, Santa Cruz, USA), or β-actin (1:20000, 66009-1-Ig, Proteintech, China) in TBS containing 0.2% Tween-20 (TBST) diluted overnight at 4 °C.

Techniques: Expressing