quantikine human angpt2 elisa kit  (R&D Systems)

Journal: Scientific Reports

Article Title: Multi-omics analysis of macrophage-associated receptor and ligand reveals a strong prognostic signature and subtypes in hepatocellular carcinoma

doi: 10.1038/s41598-024-62668-x

Figure Lengend Snippet: Mutation landscape analysis and tissue expression of core genes. ( A , B ) Mutation gene analysis based on whole exome sequencing data in patients with two subtypes of high and low risk. TCGA-LIHC cohort ( A ), GEO cohort (B). ( C ) Mutation analysis of three core genes. ( D ) CNV analysis of three core genes. ( E ) Differential expression analysis of three core genes in the TCGA-LIHC cohort in cancer and para cancer. ( F–H ) Validation of NCL ( F ), SPP1 ( G ), and ANGPT2 ( H ) expression in HCC based on the human protein atlas database.

Article Snippet: The levels of NCL (Mlbio, ml038241), IL-10 (Mlbio, ml064299), OPN (Mlbio, ml038278), and ANGPT2 (Multi Sciences, EK1215) were measured using ELISA kits according to the manufacturer’s protocol.

Techniques: Mutagenesis, Expressing, Sequencing, Quantitative Proteomics, Biomarker Discovery

Journal: Scientific Reports

Article Title: Multi-omics analysis of macrophage-associated receptor and ligand reveals a strong prognostic signature and subtypes in hepatocellular carcinoma

doi: 10.1038/s41598-024-62668-x

Figure Lengend Snippet: Correlation between signature genes and TME markers in ICI treatment. ( A – D ) The levels of OPN ( A ), ANGPT2 ( B ), NCL ( C ), and IL-10 ( D ) in the peripheral blood of patients in the ICI response and ICI resistance groups were measured and then analyzed for differences. (student's t-test. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001). ( E – G ) Correlation analysis of IL-10 and OPN ( E ), ANGPT2 ( F ), and NCL ( G ) expression levels in peripheral blood of patients with ICI resistance. (Pearson correlation analysis).

Article Snippet: The levels of NCL (Mlbio, ml038241), IL-10 (Mlbio, ml064299), OPN (Mlbio, ml038278), and ANGPT2 (Multi Sciences, EK1215) were measured using ELISA kits according to the manufacturer’s protocol.

Techniques: Expressing

Journal: Scientific Reports

Article Title: Multi-omics analysis of macrophage-associated receptor and ligand reveals a strong prognostic signature and subtypes in hepatocellular carcinoma

doi: 10.1038/s41598-024-62668-x

Figure Lengend Snippet: Expression levels of SPP1, ANGPT2, and NCL across different cell types. ( A – E ) The scatter plot demonstrates clustering and grouping results of cells within the LIHC_GSE140228_10X ( A ) and LIHC GSE140228 Smartseq2 ( E ). The distribution and expression of target genes SPP1 ( B – F ), ANGPT2 ( C – G ), and NCL ( D – H ) across different cell types.

Article Snippet: The levels of NCL (Mlbio, ml038241), IL-10 (Mlbio, ml064299), OPN (Mlbio, ml038278), and ANGPT2 (Multi Sciences, EK1215) were measured using ELISA kits according to the manufacturer’s protocol.

Techniques: Expressing

Journal: Scientific Reports

Article Title: Multi-omics analysis of macrophage-associated receptor and ligand reveals a strong prognostic signature and subtypes in hepatocellular carcinoma

doi: 10.1038/s41598-024-62668-x

Figure Lengend Snippet: Drug–gene interaction and molecular docking analyses. ( A – C ) Drug–gene interaction network of SPP1 ( A ), ANGPT2 ( B ), and NCL ( C ). ( D – F ) Molecular docking between three genes and three drugs. SPP1 and Wortmannin ( D ). ANGPT2 and Ribavirin ( E ). NCL and Itarnafloxin ( F ).

Article Snippet: The levels of NCL (Mlbio, ml038241), IL-10 (Mlbio, ml064299), OPN (Mlbio, ml038278), and ANGPT2 (Multi Sciences, EK1215) were measured using ELISA kits according to the manufacturer’s protocol.

Techniques:

Journal: The Journal of Clinical Investigation

Article Title: Cathepsin K cleavage of angiopoietin-2 creates detrimental Tie2 antagonist fragments in sepsis

doi: 10.1172/JCI174135

Figure Lengend Snippet: ( A ) Schematic representation of experiments evaluating the effects of macrophages’ conditioned media (CM-Mq) on HUVECs. ECIS, electric cell-substrate impedance sensing. ( B ) Transendothelial electrical resistance in confluent HUVEC monolayers treated with CM-Mq for 20 hours ( n = 3 per group, ANOVA with multiple comparison P values; individual resistance curves in ). ( C – E ) HUVECs stimulated with CM-Mq Veh or CM-Mq LPS for the indicated time. ANGPT2 mRNA levels measured by ( C ) RT-qPCR on HUVEC homogenates, and ANGPT2 protein level in CM measured by ( D ) ELISA or ( E ) Western analysis ( n = 3 per group, respectively). Black arrows to putative processed versions of ANGPT2. The green arrow points to putative ANGPT2 443 isoform. An uncropped image, including 0-hour stimulation with CM-Mq Veh or CM-Mq LPS , is shown in . ( F ) Schematic representation of experiments to test if CM-Mq LPS induces cell-free posttranslational modification on ANGPT2. ( G ) RAW264.7 cells were treated with LPS and concomitant application of recombinant ANGPT2 for the indicated time, after which ANGPT2 in CM was analyzed by Western blotting. Three arrowheads (blue, yellow, and purple) indicate putative processed versions of ANGPT2. ( H ) Incubation of recombinant ANGPT2 with CM-Mq LPS in the absence of cells for 37°C for 24 hours prior to Western blot analysis. Western analysis images are representative of at least 3 independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001 by Mann-Whitney except as noted.

Article Snippet: Recombinant TNF-α and mouse and human ANGPT2 were purchased from R&D Systems.

Techniques: Electric Cell-substrate Impedance Sensing, Comparison, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Western Blot, Modification, Recombinant, Incubation, MANN-WHITNEY

Journal: The Journal of Clinical Investigation

Article Title: Cathepsin K cleavage of angiopoietin-2 creates detrimental Tie2 antagonist fragments in sepsis

doi: 10.1172/JCI174135

Figure Lengend Snippet: ( A ) Comparison of polyclonal ANGPT2 antibody versus monoclonal C-terminal–specific His-tag antibody in the detection of bands of ANGPT2 incubated with CM-Mq LPS for 24 hours. ( B ) Incubation of murine Angpt2 with CM-Mq LPS at 37°C for 24 hours before Western analysis. ( C ) In silico analysis of murine and human ANGPT2 with indicated predicted cleavage sites between amino acids 50 and 80 and between amino acids 240 and 260. ( D ) Western analysis of condition media from HEK293 cells that were transfected with full-length ANGPT2 and partial ANGPT2s (55S–496F and 253E–496F). ( E and F ) Mass spectrometry analysis of 2 ANGPT2 polypeptides: 42S–71L ( E ) and 245Q–260T ( F ) incubated in the presence (bottom) or absence (top) of recombinant CATK. The peaks were identified as (peak 1) SYTFLLPEMDNCRSSSSPYVSNAVQRDAPL (3,348 Da, amino acids 42S–71L), (peak 2) SYTFLLPEMDNC (1,433 Da), (peak 3) SSSSPYVSNAVQRDAPL (1,778 Da), (peak 4) MDNCRSSSSPYVSNAVQRDAPL (2,397 Da), (peak 5) LPEMDNCRSSSSPYVSNAVQRDAPL (2,737 Da), (peak 6) SYTFLLPE (969 Da), (peak 7) SYTFLLPEMDNCR (1,589 Da), (peak 8) FLLPEMDNCRSSSSPYVSNAVQRDAPL (2,997 Da), (peak 9) SYTFLLPEMDNCRSSSSPYVSNAVQ (2,796 Da), (peak 10) QKQQHDLMETVNNLLT (1,912 Da, amino acids 245Q–260T), and (peak 11) ETVNNLLT (903 Da). The peak at 4.9 minutes in the top of F is presumably a product from incomplete synthesis that is present in the absence of CATK (1,956 Da). Recombinant CATK cleaved ANGPT2 polypeptides at the following ANGPT2 amino acids: 44T, 46L, 49E, 53C, 54R, 66Q, and 252M. ( G ) Western analysis of ANGPT2 (full length) and partial ANGPT2s (55S–496F and 253E–496F) treated with CATK and Deglycosylation Mix (NEB) enzymes. Left arrows (yellow, purple) indicated deglycosylated cleaved ANGPT2 proteins. ( H ) Schematic protein structures of ANGPT2 and 2 cleaved ANGPT2s, cANGPT2 50 and cANGPT2 25 . Predicted CATK cleavage sites were located at 54R and 252M. Western analysis images are representative of at least 3 independent experiments.

Article Snippet: Recombinant TNF-α and mouse and human ANGPT2 were purchased from R&D Systems.

Techniques: Comparison, Incubation, Western Blot, In Silico, Transfection, Mass Spectrometry, Recombinant

Journal: The Journal of Clinical Investigation

Article Title: Cathepsin K cleavage of angiopoietin-2 creates detrimental Tie2 antagonist fragments in sepsis

doi: 10.1172/JCI174135

Figure Lengend Snippet: ( A ) RAW264.7 cells pretreated with vehicle or increasing concentrations of CATK inhibitor odanacatib (ODN, from left 12.5 nM, 125 nM, 1.25 μM, 12.5 μM). IC 50 = 108 nM for mouse Catk . RAW264.7 cells were stimulated with LPS and then incubated with recombinant ANGPT2. ODN was added to the cells 1 hour prior to LPS and ANGPT2. ( B ) Incubation of recombinant ANGPT2 and CM-Mq LPS harvested from CRISPR-mediated Catk-KO RAW264.7 clonal cell lines and isogenic Cas9 control cell lines. CRISPR-mediated targeting strategy and Catk-KO scores of the 3 cell lines are described in . ( C ) Incubation of recombinant CATK and conditioned media of HEK293 cells transfected with wild-type or mutant Angpt2 expression vectors at predicted Catk cleavage sites for cANGPT2 50 , for cANGPT2 25 , or for both sites (Double). ( D ) Left: Western analysis under reduced conditions (10% β-mercaptoethanol) of CM-Mq LPS versus CM-Mq Veh incubated with recombinant ANGPT2. Arrows indicate cANGPT2 50 (orange) and cANGPT2 25 (violet). Right: Same samples as shown to the left applied to SDS-PAGE followed by Western analysis under nonreducing conditions. Predicted oligomeric status is indicated. Following LPS treatment, the 150 kDa band may indicate heterotetramerization of 2 cANGPT2 50 and 2 cANGPT2 25 monomers. ( E ) Western analysis of phospho-AKT and total AKT in HUVECs stimulated with different recombinant versions of cleaved ANGPT2 protein (1,000 ng/mL). All samples were loaded on the same gel but were noncontiguous. ( F ) Western analysis of phospho-AKT and total AKT in HUVECs stimulated with ANGPT1 (80 ng/mL) and CM of control empty vector (EV) or cANGPT2-expressing HEK293 cells. Western analysis images are representative of at least 3 independent experiments.

Article Snippet: Recombinant TNF-α and mouse and human ANGPT2 were purchased from R&D Systems.

Techniques: Incubation, Recombinant, CRISPR, Control, Transfection, Mutagenesis, Expressing, Western Blot, SDS Page, Plasmid Preparation

Journal: The Journal of Clinical Investigation

Article Title: Cathepsin K cleavage of angiopoietin-2 creates detrimental Tie2 antagonist fragments in sepsis

doi: 10.1172/JCI174135

Figure Lengend Snippet: Summary of ANGPT2/cleaved ANGPT2 proteins characteristics

Article Snippet: Recombinant TNF-α and mouse and human ANGPT2 were purchased from R&D Systems.

Techniques:

Journal: The Journal of Clinical Investigation

Article Title: Cathepsin K cleavage of angiopoietin-2 creates detrimental Tie2 antagonist fragments in sepsis

doi: 10.1172/JCI174135

Figure Lengend Snippet: ( A and B ) Circulating Catk ( A ) and Angpt2 ( B ) protein levels 24 hours after LPS (10 mg/kg) administration ( n = 8 mice per group). ( C ) Catk activity in the lungs of mice harvested 24 hours after LPS (10 mg/kg) administration ( n = 10 mice per group). ( D ) Effect of ODN (20 mg/kg, 1 hour prior to LPS administration) on lung Catk activity 24 hours after LPS (10 mg/kg) administration ( n = 5 mice per group). ( E ) Blinded scoring of histological lung injury as described in Methods. Representative images of H&E-stained lung sections are shown in , H and I. ( F ) Effect of ODN (20 mg/kg) on kidney Catk activity 24 hours after LPS (10 mg/kg) administration ( n = 5 mice per group). ( G–I ) Markers of kidney injury: ( G ) whole-kidney Lcn2 mRNA; ( H ) serum creatinine; and ( I ) blood urea nitrogen (BUN). * P < 0.05, ** P < 0.01, **** P < 0.0001 by Mann-Whitney.

Article Snippet: Recombinant TNF-α and mouse and human ANGPT2 were purchased from R&D Systems.

Techniques: Activity Assay, Staining, MANN-WHITNEY

Journal: The Journal of Clinical Investigation

Article Title: Cathepsin K cleavage of angiopoietin-2 creates detrimental Tie2 antagonist fragments in sepsis

doi: 10.1172/JCI174135

Figure Lengend Snippet: ( A and B ) Survival curves for mice after ( A ) LPS administration (10 mg/kg) or ( B ) cecal ligation puncture (CLP). Vehicle or ODN (20 mg/kg) was injected i.p. 1 hour prior to LPS administration or CLP surgery. ( C – F ) Survival curves after hydrodynamic gene transfer of indicated plasmids (10 μg DNA in 10% saline based on body weight, 4 hours prior to vehicle or ODN). Vehicle or ODN (20 mg/kg, i.p.) was administered 1 hour prior to LPS (10 mg/kg) injection. LPS injection time was recorded as time 0. Empty vector (EV) + ODN was used as the control group. ( G and H ) Survival curves for Angpt2 heterozygous or littermate wild-type control mice after ODN and LPS injection performed, as per A . The Angpt2+/+ + Veh group was used as the control group. Numbers in parentheses indicate mice per group. * P < 0.05, ** P < 0.01 by log-rank test.

Article Snippet: Recombinant TNF-α and mouse and human ANGPT2 were purchased from R&D Systems.

Techniques: Ligation, Injection, Saline, Plasmid Preparation, Control

Journal: The Journal of Clinical Investigation

Article Title: Cathepsin K cleavage of angiopoietin-2 creates detrimental Tie2 antagonist fragments in sepsis

doi: 10.1172/JCI174135

Figure Lengend Snippet: ( A and B ) Western analysis of molecular weight column filtration (cut off 50 kDa) to separate full-length ANGPT2 and cANGPT2 25 . ( A ) His-tagged recombinant ANGPT2 was incubated with CM-Mq LPS and separated into a >50 kDa and a <50 kDa fraction. ANGPT2 and cANGPT2 25 were visualized with anti-His antibody. Fraction concentration factor was calculated by determining the weight of retentate (18.7 mg) and filtrate (75.1 mg) versus prefilter weight (99 mg). Representative blot of 3 independent experiments depicted, along with ( B ) quantified band signal intensities corrected by concentration factors and normalized to prefiltration band intensity set to 100% (mean ± SD, n = 3): >50 kDa ANGPT2, 97.27% ± 9.17%; >50 kDa cANGPT2 25 , 72.19% ± 5.40%; <50 kDa ANGPT2, 0%; <50 kDa cANGPT2 25, 20.37% ± 2.30%; and cANGPT2 25 detected in both fractions combined, 92.56% ± 7.49%). ( C ) cANGPT2 25 (ng/mL) in sera of healthy individuals acting as controls (healthy, n = 10), patients in the ICU with a primary diagnosis of acute respiratory distress syndrome (ARDS, n = 20), ICU patients with a primary diagnosis of ARDS and sepsis (ARDS & Sepsis, n = 10), and ICU patients with a primary diagnosis of sepsis (Sepsis, n = 10) (Kruskal-Wallis, **P < 0.01, ***P < 0.001). ( D ) cANGPT2 25 (ng/mL) stratified by survival ( n = 31) versus death ( n = 9) 28 days after diagnosis of ARDS, ARDS and sepsis, or sepsis (Mann Whitney, *P < 0.01). ( E ) Spearman’s correlation plot showing ICU patients with diagnoses of ARDS, ARDS and sepsis, or sepsis ( n = 40) comparing major parameters from blood tests and other assessments, as defined against absolute concentration of cANGPT2 25 (ng/mL) and the ratio of cANGPT2 25 to the total amount of ANGPT2. Color indicates strength of association by correlation coefficient. ICU LOS, ICU length of stay; Pmax, peak inspiratory pressure; PEEP, peak end-expiratory pressure; SOFA, sequential organ failure assessment score; INR, international normalized ratio measuring blood coagulation; PCT, procalcitonin; LDH, lactate dehydrogenase. ( F and G ) Correlation between cANGPT2 25 (ng/mL) in ICU patients and SOFA score ( F ) and total ANGPT2 (ng/mL). Data in G are from E . Triangles indicate death within 28 days of diagnosis.

Article Snippet: Recombinant TNF-α and mouse and human ANGPT2 were purchased from R&D Systems.

Techniques: Western Blot, Molecular Weight, Filtration, Recombinant, Incubation, Concentration Assay, Biomarker Discovery, MANN-WHITNEY, Coagulation