goat anti tie2  (R&D Systems)

Journal: Nature cardiovascular research

Article Title: Angiopoietin-TIE2 feedforward circuit promotes PIK3CA-driven venous malformations.

doi: 10.1038/s44161-025-00655-9

Figure Lengend Snippet: Fig. 6 | Increased TIE2 phosphorylation and SMC coverage in Pik3ca-driven VM in mice. a, PLA staining of activated TIE2 on ear skin paraffin sections from Pik3caH1047R;Vegfr1-CreERT2 and Cre− littermate Ctrl mice, detected using pTyr and TIE2 antibodies. DAPI marks cell nuclei. b, Quantification of PLA signals within PECAM1+ blood vessels. Data represent the number of PLA dots per μm2 of PECAM1+ vessel area, mean ± s.d. (n = 8 (Ctrl) and n = 26 (Pik3caH1047R) vessels from four mice per genotype, unpaired two-tailed Student’s t-test, ****P = 0.0000054). c, Immunofluorescence of ear skin paraffin sections from Pik3caH1047R;Vegfr1- CreERT2 and Cre− littermate Ctrl mice using phospho-TIE2 antibodies. d, Phospho- TIE2 signal within EMCN+ vessels, represented as corrected total cell fluorescence (CTFC) of EMCN+ vessel area. Data points represent pTIE2 CTFC, mean ± s.d. (n = 20 (Ctrl) and n = 31 (Pik3caH1047R) vessels from two mice per genotype, unpaired two-tailed Student’s t-test, ****P = 0.000096; Extended Data Fig. 8d). e, Whole-mount immunofluorescence of ear skin from Pik3caH1047R;

Article Snippet: Human paraffin sections (5 μm) were treated as described above and antibodies used were rabbit pTyr (P-Tyr-1000 MultiMab, Cell Signaling, 8954, dilution 1:200) and goat anti-human TIE2 (R&D Systems, AF313, dilution 1:200).

Techniques: Phospho-proteomics, Staining, Two Tailed Test, Immunofluorescence, Fluorescence

Journal: Nature cardiovascular research

Article Title: Angiopoietin-TIE2 feedforward circuit promotes PIK3CA-driven venous malformations.

doi: 10.1038/s44161-025-00655-9

Figure Lengend Snippet: Fig. 7 | Increased TIE2 phosphorylation in human PIK3CA-driven VMs. a, H&E-stained paraffin sections of cutaneous VMs from individuals with PIK3CA (top) or TEK (below) mutations. Areas defined as non-lesional (NL) and VM lesions are indicated. b, PLA staining of activated TIE2, detected using pTyr and TIE2 antibodies, in representative vessels from indicated NL and VM regions. DAPI marks cell nuclei. c, Quantification of PLA signals within PECAM1+ veins, represented as mean PLA dots per EC nucleus ± s.d. (n = 7 (PIK3CA), n = 6 (TEK), n = 2 (Ctrl) and n = 5 (NL) individuals, with symbols indicating different mutations; ordinary one-way analysis of variance (ANOVA) and Tukey’s multiple-comparison test, **P(PIK3CA VM versus Ctrl vein) = 0.0027 and **P(TEK VM versus Ctrl vein) = 0.0078; Extended Data Fig. 7). d, Representative immunofluorescence image (left) and quantification (right) showing the

Article Snippet: Human paraffin sections (5 μm) were treated as described above and antibodies used were rabbit pTyr (P-Tyr-1000 MultiMab, Cell Signaling, 8954, dilution 1:200) and goat anti-human TIE2 (R&D Systems, AF313, dilution 1:200).

Techniques: Phospho-proteomics, Staining, Comparison, Immunofluorescence

Journal: Nature cardiovascular research

Article Title: Angiopoietin-TIE2 feedforward circuit promotes PIK3CA-driven venous malformations.

doi: 10.1038/s44161-025-00655-9

Figure Lengend Snippet: Fig. 6 | Increased TIE2 phosphorylation and SMC coverage in Pik3ca-driven VM in mice. a, PLA staining of activated TIE2 on ear skin paraffin sections from Pik3caH1047R;Vegfr1-CreERT2 and Cre− littermate Ctrl mice, detected using pTyr and TIE2 antibodies. DAPI marks cell nuclei. b, Quantification of PLA signals within PECAM1+ blood vessels. Data represent the number of PLA dots per μm2 of PECAM1+ vessel area, mean ± s.d. (n = 8 (Ctrl) and n = 26 (Pik3caH1047R) vessels from four mice per genotype, unpaired two-tailed Student’s t-test, ****P = 0.0000054). c, Immunofluorescence of ear skin paraffin sections from Pik3caH1047R;Vegfr1- CreERT2 and Cre− littermate Ctrl mice using phospho-TIE2 antibodies. d, Phospho- TIE2 signal within EMCN+ vessels, represented as corrected total cell fluorescence (CTFC) of EMCN+ vessel area. Data points represent pTIE2 CTFC, mean ± s.d. (n = 20 (Ctrl) and n = 31 (Pik3caH1047R) vessels from two mice per genotype, unpaired two-tailed Student’s t-test, ****P = 0.000096; Extended Data Fig. 8d). e, Whole-mount immunofluorescence of ear skin from Pik3caH1047R;

Article Snippet: Equal amounts of total protein lysates were incubated overnight at 4 °C with 1 μg of goat anti-human anti-TIE2 antibody (R&D Systems, AF313), and immunoprecipitations were performed with magnetic Protein G Dynabeads (Invitrogen, 10-003-D) for 2 h. Beads were washed three times with immunoprecipitation buffer and proteins were eluted by incubation at 95 °C for 10 min with 2× SDS sample buffer before immunoblotting analysis.

Techniques: Phospho-proteomics, Staining, Two Tailed Test, Immunofluorescence, Fluorescence

Journal: Nature cardiovascular research

Article Title: Angiopoietin-TIE2 feedforward circuit promotes PIK3CA-driven venous malformations.

doi: 10.1038/s44161-025-00655-9

Figure Lengend Snippet: Fig. 7 | Increased TIE2 phosphorylation in human PIK3CA-driven VMs. a, H&E-stained paraffin sections of cutaneous VMs from individuals with PIK3CA (top) or TEK (below) mutations. Areas defined as non-lesional (NL) and VM lesions are indicated. b, PLA staining of activated TIE2, detected using pTyr and TIE2 antibodies, in representative vessels from indicated NL and VM regions. DAPI marks cell nuclei. c, Quantification of PLA signals within PECAM1+ veins, represented as mean PLA dots per EC nucleus ± s.d. (n = 7 (PIK3CA), n = 6 (TEK), n = 2 (Ctrl) and n = 5 (NL) individuals, with symbols indicating different mutations; ordinary one-way analysis of variance (ANOVA) and Tukey’s multiple-comparison test, **P(PIK3CA VM versus Ctrl vein) = 0.0027 and **P(TEK VM versus Ctrl vein) = 0.0078; Extended Data Fig. 7). d, Representative immunofluorescence image (left) and quantification (right) showing the

Article Snippet: Equal amounts of total protein lysates were incubated overnight at 4 °C with 1 μg of goat anti-human anti-TIE2 antibody (R&D Systems, AF313), and immunoprecipitations were performed with magnetic Protein G Dynabeads (Invitrogen, 10-003-D) for 2 h. Beads were washed three times with immunoprecipitation buffer and proteins were eluted by incubation at 95 °C for 10 min with 2× SDS sample buffer before immunoblotting analysis.

Techniques: Phospho-proteomics, Staining, Comparison, Immunofluorescence

Journal: Journal of Clinical Investigation

Article Title: The mechanosensory channel PIEZO1 functions upstream of angiopoietin/TIE/FOXO1 signaling in lymphatic development

doi: 10.1172/jci176577

Figure Lengend Snippet: Figure 4. TIE signaling partially mediates AKT/FOXO1 activation triggered by PIEZO1 signaling. (A) HDLECs were transfected with siCtr, siTIE1, siTIE2, or siTIE1/siTIE2 for 48 hours and then exposed to either vehicle or 250 nM Yoda1 for 30 minutes. After fixation, cells were stained for FOXO1. This experi- ment was carried out concurrently with the one depicted in Figure 3C. The samples used in the siCtr plus DMSO and siCtr plus Yoda1 groups were identical to those in Figure 3C. Scale bar: 50 μm. (B) Quantification of cells exhibiting nuclear FOXO1 staining. This experiment was repeated 3 times. (C) HDLECs were transfected with the specified siRNAs and treated with vehicle or Yoda1 as described above. Cell lysates were subjected to Western blot analysis to assess AKT phosphorylation. TIE2 was isolated from cell lysates via immunoprecipitation and subsequently analyzed by Western blotting to evaluate its phosphorylation status. Each band represents a biological replicate sample (n = 3). (D) qPCR analysis of HDLECs transfected with siCtr or siTIE1 and subsequently treated with Yoda1 (250 nM, 24 hours) or vehicle. Expression levels of TIE1, FOXC2, GATA2, GJA4, and ITGA9 genes were measured. Data are expressed as the mean ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001, by 2-way ANOVA followed by Tukey’s multiple-comparison test (B and C) and 2-tailed, unpaired Student’s t test (D).

Article Snippet: For immunoprecipitation of TIE1 or TIE2, cell lysate was incubated with 1 μg goat anti-TIE1 antibody (R&D Systems, AF619) or goat anti-TIE2 antibody (R&D Systems, AF313) for 2 hours with rocking at 4°C.

Techniques: Activation Assay, Transfection, Staining, Western Blot, Phospho-proteomics, Isolation, Immunoprecipitation, Expressing, Comparison

Journal: Journal of Clinical Investigation

Article Title: The mechanosensory channel PIEZO1 functions upstream of angiopoietin/TIE/FOXO1 signaling in lymphatic development

doi: 10.1172/jci176577

Figure Lengend Snippet: Figure 8. Model of the PIEZO1/ANGPT/TIE/FOXO1 axis in the regu- lation of lymphatic development. Activation of the mechanosensory cation channel PIEZO1 initiates a cascade of events crucial for lymphatic development. This activation leads to an increase in intracellular calcium levels, subsequently triggering the release of ANGPT2 from intracellular vesicles and activation of the protease ADAM17. ADAM17 cleaves cell membrane–anchored TIE1, facilitating the binding and activation of TIE2 by the released ANGPT2. This activation, in turn, initiates downstream signaling through the PI3K/AKT/FOXO1 pathways. The translocation of FOXO1 from the nucleus to the cytoplasm alleviates its repression of lymphatic valve– and other lymphatic-associated genes that are crucial for lymphatic development. This finely orchestrated axis plays a pivotal role in governing the intricate processes involved in the formation and maturation of the lymphatic system.

Article Snippet: For immunoprecipitation of TIE1 or TIE2, cell lysate was incubated with 1 μg goat anti-TIE1 antibody (R&D Systems, AF619) or goat anti-TIE2 antibody (R&D Systems, AF313) for 2 hours with rocking at 4°C.

Techniques: Activation Assay, Membrane, Binding Assay, Translocation Assay