quantikine human angpt2 elisa kit  (R&D Systems)

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 5. PIEZO1 activation induces ANGPT2 exocytosis in LECs. (A) Immunostaining for ANGPT2 and FOXO1 in HDLECs transfected with siCtr or siANGPT2 and treated with DMSO or Yoda1 (250 nM, 30 minutes). Scale bar: 50 μm. (B) Western blot analysis of ANGPT2 expression in lysates from HDLECs treated with Yoda1 or DMSO. ANGPT2 concentration in the HDLEC culture medium was measured by ELISA (lower right panel). Each band rep- resents a biological replicate sample (n = 3). (C) Quantification of cells exhibiting nuclear FOXO1 staining in A. This experiment was repeated 3 times, and 3 fields were counted in each group. (D) Western blot analysis of p-AKT levels in lysates from HDLECs treated with siCtr or siANGPT2 and DMSO or Yoda1. Each band represents a biological replicate sample (n = 3). (E) Skins isolated from P1 Piezo1WB–/–E18.5 pups and their littermate controls were stained for PROX1 and ANGPT2. Scale bar: 50 μm. (F) Quantification of the ANGPT2+ areas in lymphatic vessels from 3 mice in each group. (G) Western blot analysis of AKT activation following treatment with rANGPT2 or rANGPT1 at the indicated concentrations for 30 minutes. Each band represents a biological replicate sample (n = 3). (H) FOXO1 immunostaining of HDLECs treated with vehicle or rANGPT2 (600 ng/mL, 30 minutes) and quantification of cells displaying nuclear FOXO1 staining. Scale bar: 50 μm. (I) Western blot analysis of p-AKT levels under the indicated conditions, with rANGPT2 administered at 600 ng/ mL and Yoda1 at 250 nM. Each band represents a biological replicate sample (n = 3). 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 (C, D, and I) and 2-tailed, unpaired Student’s t test (B, F, and H).

Article Snippet: ANGPT2 ELISA was carried out using the human ANGPT2 Quantikine ELISA Kit (R&D Systems, DANG20) according to the manufacturer’s instructions. qRT-PCR.

Techniques: Activation Assay, Immunostaining, Transfection, Western Blot, Expressing, Concentration Assay, Enzyme-linked Immunosorbent Assay, Staining, Isolation, 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 7. Yoda1 induces TIE1 shedding and ANGPT2 exocytosis via extracellular calcium influx. (A) Intracellular calcium levels in HDLECs treated with Yoda1 or vehicle were visualized using confocal microscopy with the cell-permeable Ca2+ indicator Fluo-8 AM. Scale bar: 50 μm. (B) Quantification of Yoda1-induced calcium influx in HDLECs pretreated with varying concentrations of the calcium chelator BAPTA. RFU, relative fluorescence units; T–T0, difference between the value measured at time point (T) and the value measured immediately prior to the treatment (T0). (C) Immunostaining for FOXO1, ANGPT2, and TIE1 in HDLECs treated with either vehicle or the calcium ionophore A23187 for 30 minutes. Arrows indicate the areas at cell-cell junctions where TIE1 shedding occurred. Scale bar: 50 μm. (D) Western blot analysis of TIE1 shedding and ANGPT2 exocytosis in HDLECs treated with vehicle or A23187. Each band represents a biological replicate sample (n = 3). (E) Western blot analysis of Yoda1-triggered TIE1 shedding and ANGPT2 exocytosis in the presence or absence of BAPTA. Each band represents a biological replicate sample (n = 3). Data are expressed as the mean ± SD. **P < 0.01 and ***P < 0.001, by 2-tailed, unpaired Student’s t test (D) and 2-way ANOVA followed by Tukey’s multiple-comparison test (E).

Article Snippet: ANGPT2 ELISA was carried out using the human ANGPT2 Quantikine ELISA Kit (R&D Systems, DANG20) according to the manufacturer’s instructions. qRT-PCR.

Techniques: Confocal Microscopy, Fluorescence, Immunostaining, Western Blot, 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: ANGPT2 ELISA was carried out using the human ANGPT2 Quantikine ELISA Kit (R&D Systems, DANG20) according to the manufacturer’s instructions. qRT-PCR.

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

Journal: The 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: ( A ) Immunostaining for ANGPT2 and FOXO1 in HDLECs transfected with siCtr or siANGPT2 and treated with DMSO or Yoda1 (250 nM, 30 minutes). Scale bar: 50 μm. ( B ) Western blot analysis of ANGPT2 expression in lysates from HDLECs treated with Yoda1 or DMSO. ANGPT2 concentration in the HDLEC culture medium was measured by ELISA (lower right panel). Each band represents a biological replicate sample ( n = 3). ( C ) Quantification of cells exhibiting nuclear FOXO1 staining in A . This experiment was repeated 3 times, and 3 fields were counted in each group. ( D ) Western blot analysis of p-AKT levels in lysates from HDLECs treated with siCtr or siANGPT2 and DMSO or Yoda1. Each band represents a biological replicate sample ( n = 3). ( E ) Skins isolated from P1 Piezo1 WB–/–E18.5 pups and their littermate controls were stained for PROX1 and ANGPT2. Scale bar: 50 μm. ( F ) Quantification of the ANGPT2 + areas in lymphatic vessels from 3 mice in each group. ( G ) Western blot analysis of AKT activation following treatment with rANGPT2 or rANGPT1 at the indicated concentrations for 30 minutes. Each band represents a biological replicate sample ( n = 3). ( H ) FOXO1 immunostaining of HDLECs treated with vehicle or rANGPT2 (600 ng/mL, 30 minutes) and quantification of cells displaying nuclear FOXO1 staining. Scale bar: 50 μm. ( I ) Western blot analysis of p-AKT levels under the indicated conditions, with rANGPT2 administered at 600 ng/mL and Yoda1 at 250 nM. Each band represents a biological replicate sample ( n = 3). 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 ( C , D , and I ) and 2-tailed, unpaired Student’s t test ( B , F , and H ).

Article Snippet: ANGPT2 ELISA was carried out using the human ANGPT2 Quantikine ELISA Kit (R&D Systems, DANG20) according to the manufacturer’s instructions.

Techniques: Immunostaining, Transfection, Western Blot, Expressing, Concentration Assay, Enzyme-linked Immunosorbent Assay, Staining, Isolation, Activation Assay, Comparison

Journal: The 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: ( A ) Intracellular calcium levels in HDLECs treated with Yoda1 or vehicle were visualized using confocal microscopy with the cell-permeable Ca 2+ indicator Fluo-8 AM. Scale bar: 50 μm. ( B ) Quantification of Yoda1-induced calcium influx in HDLECs pretreated with varying concentrations of the calcium chelator BAPTA. RFU, relative fluorescence units; T–T 0 , difference between the value measured at time point (T) and the value measured immediately prior to the treatment (T 0 ). ( C ) Immunostaining for FOXO1, ANGPT2, and TIE1 in HDLECs treated with either vehicle or the calcium ionophore A23187 for 30 minutes. Arrows indicate the areas at cell-cell junctions where TIE1 shedding occurred. Scale bar: 50 μm. ( D ) Western blot analysis of TIE1 shedding and ANGPT2 exocytosis in HDLECs treated with vehicle or A23187. Each band represents a biological replicate sample ( n = 3). ( E ) Western blot analysis of Yoda1-triggered TIE1 shedding and ANGPT2 exocytosis in the presence or absence of BAPTA. Each band represents a biological replicate sample ( n = 3). Data are expressed as the mean ± SD. ** P < 0.01 and *** P < 0.001, by 2-tailed, unpaired Student’s t test ( D ) and 2-way ANOVA followed by Tukey’s multiple-comparison test ( E ).

Article Snippet: ANGPT2 ELISA was carried out using the human ANGPT2 Quantikine ELISA Kit (R&D Systems, DANG20) according to the manufacturer’s instructions.

Techniques: Confocal Microscopy, Fluorescence, Immunostaining, Western Blot, Comparison

Journal: The 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: 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: ANGPT2 ELISA was carried out using the human ANGPT2 Quantikine ELISA Kit (R&D Systems, DANG20) according to the manufacturer’s instructions.

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