goat anti tie2  (R&D Systems)

Journal: The Journal of Clinical Investigation

Article Title: TIE2 activation by antibody-clustered endogenous angiopoietin-2 prevents capillary loss and fibrosis in experimental kidney disease

doi: 10.1172/JCI190286

Figure Lengend Snippet: ( A ) Schematic diagram of the experimental setup with UUO, contralateral control kidney (CL), and ABTAA (or IgG) administration. ( B and C ) Schematic diagram of ABTAA function ( B ) and inducible conditional mouse lines used in the study ( C ). TAM, tamoxifen induction. ( D and E ) pTIE2/TIE2 in 3-day UUO kidneys in ABTAA (ABT) and Veptp iECKO (Ve KO ) mice, respectively. Data are based on n = 3–10 mice/group and 3 ( D ) and 1 ( E ) blots (see ). ( F ) VEPTP in kidney lysates from CL and 3-day UUO kidneys in WT and Veptp iECKO mice. Data are based on n = 3 mice/group and 1 blot (see ). ( G ) TIE2 protein measured by ELISA in kidney homogenates from CL and 3-day UUO kidneys in Tie2 iECKO (T2 KO ) mice. Data are based on n = 3–5 mice/group. ( H ) Renal perfusion measured with ultrasound contrast imaging in 2-day UUO/CL kidneys from ABTAA-treated, Veptp iECKO , Tie2 iECKO , and Pdgfb iKO (Pb KO ) mice and their respective controls. The same group of sham-operated mice was used to establish baseline perfusion levels for comparison with UUO-injured kidneys. Data are based on n = 5–8 mice/group. ( I – K ) Immunohistochemistry and quantification from renal cortex for endothelial markers (endomucin [EMCN] and podocalyxin [PODXL]) in 3-day UUO kidneys from ABTAA-treated, Veptp iECKO , Tie2 iECKO , and Pdgfb iKO mice and their controls. Data are based on n = 5–11 mice/group and >600 images/marker. Scale bars: 50 μm. Data represent mean ± SD, and each symbol represents 1 mouse (females, magenta; males, cyan). * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001, determined by 1-way ANOVA and Tukey’s post hoc test ( F – H , J , and K ) or unpaired 2-tailed Student’s t test ( D and E ).

Article Snippet: The membranes were stripped and reprobed with goat anti-mouse TIE2 Ab (AF762; R&D Systems).

Techniques: Control, Enzyme-linked Immunosorbent Assay, Imaging, Comparison, Immunohistochemistry, Marker

Journal: The Journal of Clinical Investigation

Article Title: TIE2 activation by antibody-clustered endogenous angiopoietin-2 prevents capillary loss and fibrosis in experimental kidney disease

doi: 10.1172/JCI190286

Figure Lengend Snippet: ( A and B ) Semiquantitative grading of capillary length with fenestrations from micrographs of peritubular capillaries in 3-day UUO kidneys from ABTAA-treated (ABT), Veptp iECKO (Ve KO ), and Tie2 iECKO (T2 KO ) mice. Data are based on n = 4–5 mice/group and scoring of 892 micrographs. Scoring is based on percentage of endothelium with fenestrations as follows: 0, 0%–5%; 1, 6%–25%; 2, 26%–50%; 3, 51%–75%; and 4, 76%–100%. The fenestrated endothelium is indicated by an arrow. Scale bars: 2 μm. ( C ) Quantification of endothelial nuclei in Tie2 iECKO and WT mice from 1-, 3-, and 10-day UUO kidneys. Data are based on n = 4–7 mice/group, and 310 images were quantified. ( D ) Representative image with immunohistochemistry for endomucin (cyan) together with the endothelial Cdh5 -Tdtomato lineage tracer (magenta). Scale bars: 50 μm. Data represent mean ± SD, and each symbol represents 1 mouse (females, magenta; males, cyan). * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001, determined by 1-way ANOVA and Tukey’s post hoc test ( B and C ).

Article Snippet: The membranes were stripped and reprobed with goat anti-mouse TIE2 Ab (AF762; R&D Systems).

Techniques: Immunohistochemistry

Journal: The Journal of Clinical Investigation

Article Title: TIE2 activation by antibody-clustered endogenous angiopoietin-2 prevents capillary loss and fibrosis in experimental kidney disease

doi: 10.1172/JCI190286

Figure Lengend Snippet: ( A – C ) Immunohistochemistry and quantifications from renal cortex for fibrosis markers (aSMA and vimentin [VIM]) in 3-day UUO kidneys from ABTAA-treated (ABT), Veptp iECKO (Ve KO ), Tie2 iECKO (T2 KO ), and Pdgfb iKO (Pb KO ) mice and their controls. Data are based on n = 5–11 mice/group and quantifications from >600 images/marker. Scale bars: 50 μm. ( D ) Renal Col1a1 expression in ABTAA-treated mice. Data are based on n = 6 mice/group. ( E ) Renal Pdgfrb expression in Pdgfb iKO mice. Data are based on n = 5–6 mice/group. ( F ) Gene expression of Col1a1 , Tagln , and Fn1 in 3-day UUO kidneys from Tie2 iECKO mice. Data are based in n = 5–6 mice/group. Data represent mean ± SD, and each symbol represents 1 mouse (females, magenta; males, cyan). * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001, determined by 1-way ANOVA and Tukey’s post hoc test ( B – F ).

Article Snippet: The membranes were stripped and reprobed with goat anti-mouse TIE2 Ab (AF762; R&D Systems).

Techniques: Immunohistochemistry, Marker, Expressing, Gene Expression

Journal: The Journal of Clinical Investigation

Article Title: TIE2 activation by antibody-clustered endogenous angiopoietin-2 prevents capillary loss and fibrosis in experimental kidney disease

doi: 10.1172/JCI190286

Figure Lengend Snippet: ( A ) Breeding strategy for Tie2 iECKO mice with endothelial lineage tracer and myofibroblast reporter. ( B and C ) Imaging and quantification of Pdgfra -H2BGFP + nuclei (cyan) in renal cortex of 3- and 10-day UUO kidneys in WT and Tie2 iECKO (T2 KO ) mice. Data are based on n = 3–6 mice/group and quantifications from >180 images. Scale bars: 50 μm. ( D ) Representative confocal image stack (30 μm) from renal cortex showing Cdh5 -TdTomato (magenta) and Pdgfra -H2BGFP (cyan) in renal cortex 10 days after UUO in a Tie2 iECKO mouse. Scale bar: 10 μm. ( E ) Confocal images from renal cortex showing tubular epithelial marker NaK/ATPase (blue), Pdgfra -GFP (cyan), and Cdh5 -TdTomato (magenta) 10 days after UUO in WT and Tie2 iECKO mice. Representative images of n = 3 mice per group. Scale bars: 25 μm. Data in graphs represent mean ± SD, and each symbol represents 1 mouse (females, magenta; males, cyan). ** P < 0.01, *** P < 0.001, and **** P < 0.0001, determined by 1-way ANOVA and Tukey’s post hoc test ( C ).

Article Snippet: The membranes were stripped and reprobed with goat anti-mouse TIE2 Ab (AF762; R&D Systems).

Techniques: Imaging, Marker

Journal: The Journal of Clinical Investigation

Article Title: TIE2 activation by antibody-clustered endogenous angiopoietin-2 prevents capillary loss and fibrosis in experimental kidney disease

doi: 10.1172/JCI190286

Figure Lengend Snippet: ( A ) Tubular segments with pathological vacuoles (red arrow) in kidney sections stained with toluidine blue. ( B ) Quantification of tubular segments with vacuoles from ABTAA-treated (ABT), Veptp iECKO (Ve KO ), and Tie2 iECKO (T2 KO ) mice. Data are based on n = 4–5 mice/group and >10,000 tubular cross sections. ( C ) RNA-ISH for Pecam1 (cyan) and Pdgfb (magenta) in 3-day UUO kidneys. Representative image of n = 4 mice. Scale bars: 50 μm. ( D and E ) Gene expression of Kim1 , Pdgfb , Pdgfrb , Tie2 , and Angpt1 in UUO kidneys from indicated time points. Data are based on n = 3–7 mice/group. ( F ) RNA-ISH for Angpt1 (magenta), mesenchymal marker Pdgfrb (cyan), and tubular marker Atp1a1 (blue) in 3-day UUO kidneys. Representative image of n = 3 mice. Scale bars: 50 μm. ( G – I ) Expression of Pdgfb/PDGFB in 3-day UUO kidneys from ABTAA-treated, Veptp iECKO , and Pdgfb iKO (Pb KO ) mice. Data are based on n = 5–9 mice/group. ( J ) Patient data retrieved from Nephroseq for renal CDH5 (endothelial marker), ANGPT2 , PDGFB , and ANGPT1 expression in CKD and renal dysfunction compared with normal human kidney. Data in graphs ( B , D , E , and G – I ) represent mean ± SD, and each symbol represents 1 mouse (females, magenta; males, cyan). * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001, determined by 1-way ANOVA. Human data ( J ) represent Log2 expression and statistical differences from Nephroseq (see Methods).

Article Snippet: The membranes were stripped and reprobed with goat anti-mouse TIE2 Ab (AF762; R&D Systems).

Techniques: Staining, Gene Expression, Marker, Expressing

Journal: The Journal of Clinical Investigation

Article Title: TIE2 activation by antibody-clustered endogenous angiopoietin-2 prevents capillary loss and fibrosis in experimental kidney disease

doi: 10.1172/JCI190286

Figure Lengend Snippet: ( A ) Schematic diagram of administration of ABTAA for evaluation in 10-day UUO kidneys. ( B – F ) Immunohistochemistry of renal cortex for capillary density (endomucin [EMCN] and podocalyxin [PODXL]) and tubulointerstitial fibrosis (aSMA and vimentin) in 10-day UUO kidneys from ABTAA-treated mice (ABT) and Tie2 iECKO (T2 KO ) mice. Data are based on n = 6–7 mice/group and quantification of >220 images/marker. Scale bars: 50 μm. ( E ) Protein concentration for PDGFB in 10-day UUO kidneys from ABTAA-treated mice. Data are based on n = 5 mice/group. Data in graphs represent mean ± SD, and each symbol represents 1 mouse (females, magenta; males, cyan). * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001, determined by 1-way ANOVA and Tukey’s post hoc test.

Article Snippet: The membranes were stripped and reprobed with goat anti-mouse TIE2 Ab (AF762; R&D Systems).

Techniques: Immunohistochemistry, Marker, Protein Concentration

Journal: Development (Cambridge, England)

Article Title: TIE1 dependent lymphatic vascular remodeling is mediated by its second tyrosine kinase domain.

doi: 10.1242/dev.204469

Figure Lengend Snippet: Fig. 1. TIE2 and TIE1 expression in LECs of developing and mature lymphatics.

Article Snippet: Primary antibodies for immunohistochemistry Antibody Supplier Catalogue # Application Goat anti-mouse VEGFR3 R&D Systems AF743 1:50 Goat anti-human TIE1 R&D Systems AF619 1:100 Goat anti-mouse TIE2 R&D Systems AF762 1:200 Goat anti-mouse integrinα9 R&D Systems AF3827 1:50 Goat anti-VE-Cadherin R&D Systems AF1002 1:400 Goat anti-human Prox1 R&D Systems AF2727 1:100 Sheep anti-mouse FOXC2 R&D Systems AF6989 1:200 Rabbit anti-GFP Invitrogen A11122 1:200 Rabbit anti-FOXO1 Cell Signaling 2880 1:200 Rabbit anti-Lyve1 abcam Ab14917 1:800 Rabbit anti-Prox1 abcam Ab37128 1:250 Rat anti-mouse CD31 PD Pharmingen 557355 1:200 Rat anti-endomucin abcam ab106100 1:200 Development: doi:10.1242/dev.204469: Supplementary information D ev el o pm en t • S up pl em en ta ry in fo rm at io n

Techniques: Expressing

Journal: Development (Cambridge, England)

Article Title: TIE1 dependent lymphatic vascular remodeling is mediated by its second tyrosine kinase domain.

doi: 10.1242/dev.204469

Figure Lengend Snippet: Fig. 2. LEC specific deletion of Tie1 but not Tie2 results in defective lymphatic

Article Snippet: Primary antibodies for immunohistochemistry Antibody Supplier Catalogue # Application Goat anti-mouse VEGFR3 R&D Systems AF743 1:50 Goat anti-human TIE1 R&D Systems AF619 1:100 Goat anti-mouse TIE2 R&D Systems AF762 1:200 Goat anti-mouse integrinα9 R&D Systems AF3827 1:50 Goat anti-VE-Cadherin R&D Systems AF1002 1:400 Goat anti-human Prox1 R&D Systems AF2727 1:100 Sheep anti-mouse FOXC2 R&D Systems AF6989 1:200 Rabbit anti-GFP Invitrogen A11122 1:200 Rabbit anti-FOXO1 Cell Signaling 2880 1:200 Rabbit anti-Lyve1 abcam Ab14917 1:800 Rabbit anti-Prox1 abcam Ab37128 1:250 Rat anti-mouse CD31 PD Pharmingen 557355 1:200 Rat anti-endomucin abcam ab106100 1:200 Development: doi:10.1242/dev.204469: Supplementary information D ev el o pm en t • S up pl em en ta ry in fo rm at io n

Techniques:

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: Alternatively, sections were subjected to PLA using rabbit pTyr (P-Tyr-1000 MultiMab, Cell Signaling, 8954, dilution 1:200) and goat anti-mouse TIE2 (R&D Systems, AF762, dilution 1:200), followed by co-staining using hamster anti-mouse CD31/PECAM1 2H8 (Invitrogen, MA3105, 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: Alternatively, sections were subjected to PLA using rabbit pTyr (P-Tyr-1000 MultiMab, Cell Signaling, 8954, dilution 1:200) and goat anti-mouse TIE2 (R&D Systems, AF762, dilution 1:200), followed by co-staining using hamster anti-mouse CD31/PECAM1 2H8 (Invitrogen, MA3105, 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