Journal: Scientific Reports

Article Title: Angiopoietins bind thrombomodulin and inhibit its function as a thrombin cofactor

doi: 10.1038/s41598-017-18912-8

Figure Lengend Snippet: Ang1 and Ang2 interact with TM via their fibrinogen-like domains. ( a ) Ang1 and Ang2 bind saturably to COS cells overexpressing TM. Cells were incubated with varying concentrations of Ang1-FD-Fc or Ang2-FD-Fc, and binding was assessed with an alkaline phosphatase-conjugated secondary antibody. Specific binding, given in OD405 units (see Methods), is defined as the difference between binding to TM-transfected COS cells and binding to COS cells transfected with empty vector. ( b ) The soluble extracellular domains of either TM or Tie2 (100 nM) were incubated on ice for 60 minutes with BowAng1 (Ang1), BowAng2 (Ang2) or human Fc control protein at the indicated concentrations (BowAng1 and BowAng2 are chimeric angiopoietins that contain human Fc, see Methods for details). The Fc-containing proteins were then collected on protein A/G beads and co-precipitation of TM or Tie2 was assessed by western blot. ( c ) BowAng1 (Ang1) or BowAng2 (Ang2) (100 nM) were incubated with the soluble extracellular domain of TM (100 nM) in the absence or presence of the soluble extracellular domain of Tie2 (2 μM). The Fc-containing BowAng1 and BowAng2 were then collected on protein A/G beads and co-precipitation of TM and Tie2 was assessed by western blot.

Article Snippet: Wells were then washed and incubated at RT for 1 hour with 100 μl of biotinylated goat anti-Ang1 antibody (R&D Systems, catalog number BAF923) at 0.2 μg/ml.

Techniques: Incubation, Binding Assay, Transfection, Plasmid Preparation, Control, Western Blot

Journal: Scientific Reports

Article Title: Angiopoietins bind thrombomodulin and inhibit its function as a thrombin cofactor

doi: 10.1038/s41598-017-18912-8

Figure Lengend Snippet: Ang1 and Ang2 inhibit TM-dependent formation of APC and TAFIa. ( a ) HUVECs were incubated with protein C and thrombin plus either 1 μM of Fc control protein or the indicated concentrations of native Ang1 or Ang2 for 60 minutes. Cell supernatants were then assayed for APC levels. The graph depicts the relative amounts of APC formation (compared to APC formation in the presence of the Fc control protein) as a function of angiopoietin concentration. The error bars represent the SD, n = 3. ( b ) HUVECs were incubated with protein C and thrombin plus either Fc control protein (0.5 μM), native Ang1 (0.5 μM) or PF4 (10 μg/ml, ~1.3 μM) for 60 minutes and cell supernatants were then assayed for APC levels. The graph depicts the relative amounts of APC generated (the amount of APC generated in the presence of Fc control protein was assigned a value of 1.0). The error bars represent the SD, n = 3. Relative levels of APC in the Ang1 and the PF4 treatment groups were significantly different from the control group (***P < 0.001, one-way ANOVA with Tukey’s multiple comparisons test). ( c ) HUVECs were incubated with protein C and thrombin plus either 1 μM of Fc control protein or the indicated concentrations of native Ang1 or Ang2 for 60 minutes. Cell supernatants were then assayed for TAFIa levels. The graph depicts the relative amounts of TAFIa generated (the amount of TAFIa generated in the presence of Fc control protein was assigned a value of 1.0). The error bars represent the SD, n = 3. Relative levels of TAFIa in the Ang1 100 nM and the Ang2 100 nM treatment groups were significantly different from the control group (***P < 0.001, one-way ANOVA with Tukey’s multiple comparisons test). ( d , e ) COS cells overexpressing TM were incubated with the indicated concentrations of native Ang1 or Ang2 for 30 minutes, and then assayed for APC ( d ) or TAFIa ( e ) formation upon addition of thrombin plus protein C or TAFI. The graphs depict relative APC or TAFIa formation as a function of angiopoietin concentration (the amounts formed in the absence of angiopoietin were assigned a value of 1.0).

Article Snippet: Wells were then washed and incubated at RT for 1 hour with 100 μl of biotinylated goat anti-Ang1 antibody (R&D Systems, catalog number BAF923) at 0.2 μg/ml.

Techniques: Incubation, Control, Concentration Assay, Generated

Journal: Scientific Reports

Article Title: Angiopoietins bind thrombomodulin and inhibit its function as a thrombin cofactor

doi: 10.1038/s41598-017-18912-8

Figure Lengend Snippet: Inhibition of APC formation by Ang1 is independent of Tie2. HUVECs were infected with adenoviruses encoding either a control shRNA or a Tie2 specific shRNA. At 3 days after infection, cells were either lysed for western blot to demonstrate Tie2 knockdown ( a ) or used to test the effect of native Ang1 on APC formation in the presence of thrombin plus protein C ( b ). The graph depicts the relative amounts of APC generated in the presence of 1 μM Fc control protein or native Ang1 (the amount of APC generated in the presence of Fc control protein was assigned a value of 1.0). The error bars represent the SD, n = 3. Ang1 significantly inhibited APC formation in cells treated with control shRNA or Tie2 shRNA (***P < 0.001, one-way ANOVA with Tukey’s multiple comparisons test).

Article Snippet: Wells were then washed and incubated at RT for 1 hour with 100 μl of biotinylated goat anti-Ang1 antibody (R&D Systems, catalog number BAF923) at 0.2 μg/ml.

Techniques: Inhibition, Infection, Control, shRNA, Western Blot, Knockdown, Generated

Journal: Scientific Reports

Article Title: Angiopoietins bind thrombomodulin and inhibit its function as a thrombin cofactor

doi: 10.1038/s41598-017-18912-8

Figure Lengend Snippet: Ang1 and Ang2 inhibit binding of thrombin to TM. (a , b) Soluble extracellular domain of TM at 100 nM was incubated with 5 nM thrombin in the presence of the indicated concentrations of native Ang1, native Ang2 or native Ang4. Following collection of TM on Ni-NTA beads, the beads were washed and bound proteins were eluted in SDS sample buffer. Western blots were performed to assess pull-down of TM and co-precipitation of thrombin.

Article Snippet: Wells were then washed and incubated at RT for 1 hour with 100 μl of biotinylated goat anti-Ang1 antibody (R&D Systems, catalog number BAF923) at 0.2 μg/ml.

Techniques: Binding Assay, Incubation, Western Blot

Journal: Scientific Reports

Article Title: Angiopoietins bind thrombomodulin and inhibit its function as a thrombin cofactor

doi: 10.1038/s41598-017-18912-8

Figure Lengend Snippet: Ang 1 is present in platelets and is released upon activation. ( a ) Platelets were isolated from C57BL/6 mice as described in the Methods and lysed in SDS sample buffer. Aliquots of platelet lysate representing ~1.6 × 10 6 platelets were run on SDS gels along with the indicated amounts (ng) of mouse PF4, human Ang1 or human Ang2 to allow estimation of the levels of these proteins in the platelet lysate. The Ang1 and Ang2 antibodies recognize both the human and mouse proteins. ( b ) Blood was collected from C57BL/6 mice by cardiac puncture and used to prepare either plasma or serum. Ang1 and PF4 levels were then determined by ELISA as described in the Methods. Ang1 levels are given as apparent concentrations since the standard curve used to estimate Ang1 concentration was generated with human Ang1 protein. Bars represent the mean and SD, n = 4. Both Ang1 and PF4 levels were significantly higher in serum (**P < 0.01; ***P < 0.001, t -test). ( c ) C57BL/6 mice were injected via the tail vein with PBS or 10 units of thrombin. After 10 minutes, blood was collected by cardiac puncture and plasma was prepared. Plasma levels of Ang1 and PF4 were determined by ELISA. Bars represent the mean and SD (n = 9 for PBS, n = 8 for thrombin). The levels of both Ang1 and PF4 were significantly higher in the thrombin-treated samples (**P < 0.01; ***P < 0.001, t -test). ( d ) Blood was collected from C57BL/6 mice either by cardiac puncture (systemic) or by tail nick as described in the Methods section. Plasma was prepared and Ang1 and PF4 levels were determined by ELISA. Bars represent the mean and SD (n = 9 for systemic samples, n = 10 for tail nick samples). The levels of both Ang1 and PF4 were significantly higher in the tail nick samples (***P < 0.001, t -test).

Article Snippet: Wells were then washed and incubated at RT for 1 hour with 100 μl of biotinylated goat anti-Ang1 antibody (R&D Systems, catalog number BAF923) at 0.2 μg/ml.

Techniques: Activation Assay, Isolation, Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Concentration Assay, Generated, Injection

Journal: BMC cardiovascular disorders

Article Title: Left ventricular hypertrophy in young hypertensives: the possible crosstalk of mTOR and angiotensin-II -a case-control study.

doi: 10.1186/s12872-025-04470-9

Figure Lengend Snippet: Fig. 6 shows mTOR (a) and Ang II (b) in normotensive, HTN with or with out LVH. Values are represented as median and analysed by Kruskal-Wallis. *P < 0.05 vs. NORM, #<0.05 vs. HWOLVH. Ang II (Angiotensin II); mTOR (mammalian target organ of rapamycin)

Article Snippet: The following ELISA kits were used human mTOR ELISA Kit (Cat: ELK9237), human CHEM (Chemerin) ELISA Kit (Cat: ELK1953) and human GAL3 (Galectin 3) ELISA Kit from ELK Biotechnology Co. Ltd. (1312 17th Street #692 Denver, CO 80202 USA), human Ang-II (Angiotensin II) ELISA Kit from Elabscience Biotechnology Inc. (Wuhan, Hubei, P.R.C., China), human Troponin T ELISA kit produced by BT laboratories (Cat No: E2285Hu) and NF-kβ (human p50) Transcription Factor Assay Kit (Cat # KA1339 V.01) produced by ABNOVA.

Techniques:

Journal: BMC cardiovascular disorders

Article Title: Left ventricular hypertrophy in young hypertensives: the possible crosstalk of mTOR and angiotensin-II -a case-control study.

doi: 10.1186/s12872-025-04470-9

Figure Lengend Snippet: Fig. 9 shows the correlation between Ang II (a), LVM (b), LVMI (c) and mTOR in HWLVH. Ang-II (r = 0.489, p = 0.034) * #; LVM (r = 0.419, p = 0.06) β; LVMI (r = 0.210, p = 0.417) #; Ang II (Angiotensin II). βPearson correlation; #Spearman correlation; *statistically significant (p < 0.05)

Article Snippet: The following ELISA kits were used human mTOR ELISA Kit (Cat: ELK9237), human CHEM (Chemerin) ELISA Kit (Cat: ELK1953) and human GAL3 (Galectin 3) ELISA Kit from ELK Biotechnology Co. Ltd. (1312 17th Street #692 Denver, CO 80202 USA), human Ang-II (Angiotensin II) ELISA Kit from Elabscience Biotechnology Inc. (Wuhan, Hubei, P.R.C., China), human Troponin T ELISA kit produced by BT laboratories (Cat No: E2285Hu) and NF-kβ (human p50) Transcription Factor Assay Kit (Cat # KA1339 V.01) produced by ABNOVA.

Techniques:

Journal: Investigative ophthalmology & visual science

Article Title: Short-Term Hyperuricemia Leads to Structural Retinal Changes That Can be Reversed by Serum Uric Acid Lowering Agents in Mice.

doi: 10.1167/iovs.63.10.8

Figure Lengend Snippet: FIGURE 2. Hyperuricemia induction and intervention by allopurinol and benzbromarone, and ELISA measurement of serum UA and Ang II. (A) Serum UA concentrations in six study groups on day 1 and at the end of weeks 4, 6, and 8. (B) Serum angiotensin II concentrations in six study groups on day 1 and at the end of weeks 4, 6, and 8. The single star and double stars represent a significant difference compared to the control group (P < 0.05 and P < 0.01, respectively). The double pound signs represent a significant difference (P < 0.01) compared to the high-dose groups. The statistics were performed by the Kruskal-Wallis test and Wilcoxon test. Ang II, angiotensin II; ELISA, enzyme-linked immunosorbent assay; OA, oxonic acid potassium salt; UA, uric acid.

Article Snippet: The SUA and Ang II were measured using a commercially available mouse Uric Acid Colorimetric Assay Kit (cat. No. E-BC-K016-M; Elabscience Biotechnology Co., Ltd.) and Mouse Angiotensin II ELISA Kit (cat. No. E-ELM2612; Elabscience Biotechnology Co., Ltd.).

Techniques: Enzyme-linked Immunosorbent Assay, Control

Journal: Investigative ophthalmology & visual science

Article Title: Short-Term Hyperuricemia Leads to Structural Retinal Changes That Can be Reversed by Serum Uric Acid Lowering Agents in Mice.

doi: 10.1167/iovs.63.10.8

Figure Lengend Snippet: FIGURE 6. Summary of HUA-induced pathogenesis leading to early AMD or relevant retinopathy. AMD, age-related macular degenera- tion; Ang II, angiotensin II; GCL, ganglion cell layer; HUA, hyper- uricemia; OS, outer segment; RGC, retina ganglion cell; RPE, retinal pigment epithelium.

Article Snippet: The SUA and Ang II were measured using a commercially available mouse Uric Acid Colorimetric Assay Kit (cat. No. E-BC-K016-M; Elabscience Biotechnology Co., Ltd.) and Mouse Angiotensin II ELISA Kit (cat. No. E-ELM2612; Elabscience Biotechnology Co., Ltd.).

Techniques: