phospho tie2  (R&D Systems)

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

Journal: bioRxiv

Article Title: Ubiquitin ligase CHFR impairs Tie2 signaling via K 48 -linked ubiquitylation and degradation of Akt1 in endothelial cells

doi: 10.64898/2026.03.31.715582

Figure Lengend Snippet: a HMEC were transfected with scrambled-siRNA (sc-siRNA) or CHFR-siRNA. At 72 h post-transfection, cells stimulated with rAng1 (400 ng/ml) for different time intervals were used for IB analysis to determine phosphorylation of Akt1 at S473 and T308 ( n = 3 independent experiments). b Control HMEC and CHFR knockdown HMEC stimulated with rAng1 as above were stained with phospho-473-Akt1 specific antibody. c Control HMEC and CHFR knockdown HMEC were transfected with the Akt1 biosensor plasmid and then stimulated with rAng1 as above to assess live cell Akt1 activity by measuring the FRET ratio ( p <0.0001) ( left panel shows basal Akt activity; right panel shows Akt activity in response to rAng1 challenge). d-f Control HMEC and CHFR-siRNA treated HMEC stimulated with rAng1 were used for IB analysis to determine tyrosine phosphorylation Tie2 ( d ), phosphorylation of GSK3β at S9 and phosphorylation of β-catenin ( e ), ( n = 3 independent experiments). a, d-e Shown are mean values ± SEM ( n = 3 independent experiments; two-way ANOVA followed by Tukey’s post-hoc test).

Article Snippet: Rabbit polyclonal antibody (pAb) against phospho-Tie2 (catalog #AF3909; IB, 1:1000), goat polyclonal antibody (pAb) against PDGFRβ (catalog #AF1042; IS, 1:100) were from R&D system.

Techniques: Transfection, Phospho-proteomics, Control, Knockdown, Staining, Plasmid Preparation, Activity Assay

Journal: bioRxiv

Article Title: Ubiquitin ligase CHFR impairs Tie2 signaling via K 48 -linked ubiquitylation and degradation of Akt1 in endothelial cells

doi: 10.64898/2026.03.31.715582

Figure Lengend Snippet: a HMEC were transfected with WT or K/R-mutant Akt1 constructs. At 36 h after transfection, cells were treated with LPS (5 μg/ml) for 0, 12, and 24h and then cells were used for IB to determine expression of Akt1 and VE-cadherin. Shown are mean values ± SEM (n = 3 independent experiments; two-way ANOVA followed by Tukey’s post hoc test). b TIME endothelial cells (telomerase-immortalized human dermal microvascular endothelial cell line) were transfected with WT or K/R mutant Akt1 constructs and stimulated with LPS (5 μg/ml) for 0 and 6 h. Confocal imaging showed that expression of K/R mutant Akt1 prevents degradation of VE-cadherin. c WT mice were injected (i.v.) with liposome-encapsulated pmCherry-tagged WT or K/R mutant Akt1 constructs. Lungs harvested 96 h after injection were subjected to cryosection and stained with EC marker antibody vWF (green). Confocal imaging confirms expression of pmCherry-Akt1 (red) plasmid in lung endothelial cells. d-f Liposome-mediated delivery of Akt1 (WT) or K/R-mutated Akt1 in WT mice prevents degradation of VE-cadherin, mitigates LPS-induced lung vascular leak (EBA uptake), and reduces PMN transmigration (MPO assay). Shown are mean values ± SEM ( n = 3 or n = 5 mice/group; two-way ANOVA followed by Tukey’s post hoc test). g Model for E3 ligase CHFR regulation of endothelial junctional barrier integrity. Under baseline condition, constitutive Ang1-Tie2 signaling in EC maintains endothelial junctional barrier through Akt1 activation-mediated inhibition of the transcription factor FoxO1 activation and Ang-2 expression. During vascular inflammatory conditions such as sepsis, TLR4 signaling induces the expression of E3 ligase CHFR in a FoxO1-dependent manner. Then the upregulated CHFR mediates K 48 -linked polyubiquitylation and degradation of Akt1 and VE-cadherin (Tiruppathi et al., 2023) to disassemble EC junctional barrier. CHFR-mediated loss of FoxO1 negative regulator Akt1 expression in EC leads to increased FoxO1 expression which in turn promotes sustained expression of Ang-2 in EC to induce life-threatening pulmonary edema.

Article Snippet: Rabbit polyclonal antibody (pAb) against phospho-Tie2 (catalog #AF3909; IB, 1:1000), goat polyclonal antibody (pAb) against PDGFRβ (catalog #AF1042; IS, 1:100) were from R&D system.

Techniques: Transfection, Mutagenesis, Construct, Expressing, Imaging, Injection, Staining, Marker, Plasmid Preparation, Transmigration Assay, MPO Assay, Activation Assay, Inhibition