mouse upa total antigen assay elisa kit  (Innovative Research Inc)

Journal: EMBO Molecular Medicine

Article Title: uPA‐PAI‐1 heteromerization promotes breast cancer progression by attracting tumorigenic neutrophils

doi: 10.15252/emmm.202013110

Figure Lengend Snippet: Production of different cytokines by mouse RAW macrophages upon exposure to recombinant murine uPA‐PAI‐1 heteromers or saline as assessed by multiplex ELISA, results are shown as heatmap. In addition, quantitative data are provided for the most abundantly produced cytokines (mean ± SEM for n = 5 experiments per group; # P < 0.05 vs. saline; t ‐test).

Article Snippet: After incubation for 60 min and subsequent washing of the wells three times with PBS, the amount of uPA bound to PAI‐1 in the wells was quantified employing a Mouse uPA total antigen assay ELISA kit (Molecular Innovations, Novi, Michigan, USA).

Techniques: Recombinant, Multiplex Assay, Enzyme-linked Immunosorbent Assay, Produced

Journal: EMBO Molecular Medicine

Article Title: uPA‐PAI‐1 heteromerization promotes breast cancer progression by attracting tumorigenic neutrophils

doi: 10.15252/emmm.202013110

Figure Lengend Snippet: A Correlation of uPA or PAI‐1 protein expression and neutrophil infiltration in tumors as assessed by ELISA as well as immunohistochemistry and light microscopy in human breast cancer samples (histological grade: G1), representative images (scale bar: 100 µm) and quantitative data are shown. B Correlation analyses of RNA expression levels of uPA (PLAU gene), PAI‐1 (SERPINE1 gene), and the neutrophil marker gene FPR1, and overall survival of breast cancer patients (stages 0 and 1) with high and low PLAU or SERPINE1 gene expression levels (cutoff z ≥ 2.0) in the METABRIC breast cancer cohort. C Surface expression of MMP‐9, VEGF, and NE as assessed on circulating neutrophils isolated from the peripheral blood of WT mice (saline) or from the peritoneal cavity of WT mice 6 h after intraperitoneal stimulation with uPA‐PAI‐1 (uPA‐PAI‐1) by multi‐channel flow cytometry, quantitative data are shown (mean ± SEM for n = 4–6 mice per group; # P < 0.05 vs. saline; t ‐test). D, E Proliferation of (D) 4T1 breast cancer cells or (E) bEnd.3 microvascular endothelial cells upon exposure to recombinant murine uPA‐PAI‐1, the uPA‐PAI‐1 inhibitor WX‐340, or primary neutrophils isolated from the peritoneal cavity of WT mice undergoing 6 h of intraperitoneal stimulation with uPA‐PAI‐1 with or without addition of a NE inhibitor as assessed by a MTT assay, quantitative data are shown (mean ± SEM for n = 3 experiments per group; # P < 0.05 vs. neutrophils; * P < 0.05 vs. neutrophils + vehicle / # P < 0.05 vs. 2 % FCS medium; one‐way ANOVA/ t ‐test). F Formation of NETs (histone H3 + ; green) as assessed ex vivo by confocal microscopy in the cremaster muscle of WT mice 6 h after intrascrotal injection of uPA‐PAI‐1, TNF, or saline, PECAM‐1/CD31 + postcapillary venules (blue) and Ly‐6G + neutrophils (red) are depicted. Representative images are shown (scale bar: 50 µm).

Article Snippet: After incubation for 60 min and subsequent washing of the wells three times with PBS, the amount of uPA bound to PAI‐1 in the wells was quantified employing a Mouse uPA total antigen assay ELISA kit (Molecular Innovations, Novi, Michigan, USA).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Immunohistochemistry, Light Microscopy, RNA Expression, Marker, Isolation, Flow Cytometry, Recombinant, MTT Assay, Ex Vivo, Confocal Microscopy, Injection

Journal: EMBO Molecular Medicine

Article Title: uPA‐PAI‐1 heteromerization promotes breast cancer progression by attracting tumorigenic neutrophils

doi: 10.15252/emmm.202013110

Figure Lengend Snippet: A, B Correlation of uPA or PAI‐1 protein expression (ELISA) and neutrophil infiltration (histochemistry and light microscopy) in human breast cancer samples, representative images (A; histological grades: G1‐3; scale bar: 100 µm) and quantitative data (B; histological grades: G2 or G3; n = 10–29 samples per group).

Article Snippet: After incubation for 60 min and subsequent washing of the wells three times with PBS, the amount of uPA bound to PAI‐1 in the wells was quantified employing a Mouse uPA total antigen assay ELISA kit (Molecular Innovations, Novi, Michigan, USA).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Light Microscopy

Journal: EMBO Molecular Medicine

Article Title: uPA‐PAI‐1 heteromerization promotes breast cancer progression by attracting tumorigenic neutrophils

doi: 10.15252/emmm.202013110

Figure Lengend Snippet: Effect of compound WX‐340 on binding of recombinant murine uPA to PAI‐1 protein as assessed by ELISA, quantitative data are shown (mean ± SEM for n = 3 experiments per group; # P < 0.05 vs. drug vehicle; one‐way ANOVA). Intravascular rolling and firm adherence as well as transmigration of neutrophils and classical monocytes (cMOs) to the inflamed perivascular tissue as assessed 6 h after intrascrotal injection of TNF in postcapillary venules of the cremaster muscle of WT mice, quantitative data are shown (mean ± SEM for n = 6 mice per group; # P < 0.05 vs. saline; * P < 0.05 vs. drug vehicle; one‐way ANOVA). Expression of uPA or PAI‐1 (green) in tumors as assessed in an orthotopic model of 4T1 breast cancer in WT mice by confocal microscopy, PECAM‐1/CD31 + postcapillary venules (blue) and Ly‐6G + neutrophils (red) are depicted (scale bar: 100 µm). Relative development rates and tumor weight in animals treated with WX‐340 a priori or therapeutically after 1 week after tumor cell injection on a daily basis as assessed in an orthotopic model of 4T1 breast cancer in WT mice (mean ± SEM for n = 4–6 mice per group; * P < 0.05 vs. drug vehicle; one‐way ANOVA). Intravascular rolling and firm adherence of neutrophils as assessed in the tumor microvasculature 14 days after intradermal injection of 4T1 cells in the right ear of WT mice treated with WX‐340 or vehicle by multi‐channel in vivo microscopy, quantitative data are shown (mean ± SEM for n = 4–6 mice per group; * P < 0.05 vs. drug vehicle; t ‐test). Numbers of neutrophils and 4T1 tumor cells in lungs and brain of WT mice receiving WX‐340 or vehicle therapeutically after 1 week after tumor cell injection on a daily basis as assessed 14 days after intravenous injection of 4T1 tumor cells by multi‐channel flow cytometry, quantitative data are shown (mean ± SEM for n = 4–6 mice per group; * P < 0.05 vs. drug vehicle; t ‐test).

Article Snippet: After incubation for 60 min and subsequent washing of the wells three times with PBS, the amount of uPA bound to PAI‐1 in the wells was quantified employing a Mouse uPA total antigen assay ELISA kit (Molecular Innovations, Novi, Michigan, USA).

Techniques: Binding Assay, Recombinant, Enzyme-linked Immunosorbent Assay, Transmigration Assay, Injection, Expressing, Confocal Microscopy, In Vivo, Microscopy, Flow Cytometry

Journal: Journal of Cell Science

Article Title: Urokinase-type plasminogen activator-mediated crosstalk between N-cadherin and β-catenin promotes wound healing

doi: 10.1242/jcs.255919

Figure Lengend Snippet: uPA promotes astrocytic healing via a plasminogen-independent mechanism. (A) Growth of a monolayer of Wt and Plg−/− astrocytes 24 h after a wound injury and treatment with 5 nM of uPA [n=27 (Wt) and 27 (Plg−/−)] or vehicle [control; n=37 (Wt) and 39 (Plg−/−)]. Results are presented as percentage compared to growth of Wt astrocytes treated with vehicle (control). Unpaired two-tailed Student's t-test. (B) Growth of a monolayer of Wt astrocytes 24 h after a wound injury and treatment with vehicle (control, C, n=11) or 5 nM of uPA ATF (n=14). Unpaired two-tailed Student's t-test. (C) Growth of a monolayer of Wt astrocytes 24 h after a wound injury and treatment with vehicle (control, C, n=19) or 100 nM of plasmin (n=21). Unpaired two-tailed Student's t-test. (D) Growth of a monolayer of PAI-1−/− astrocytes (n=13) 24 h after a wound injury. Results are presented as percentage compared to growth of a monolayer of astrocytes prepared from Wt littermate controls (n=22) exposed to similar experimental conditions. Unpaired two-tailed Student's t-test. In all box plots, the box represents the 25–75th percentiles, and the median is indicated. The whiskers show the range.

Article Snippet: The ELISA assay for uPA, and recombinant plasmin, murine uPA and its N-terminal fragment (ATF) were purchased from Molecular Innovations (Novi, MI; Cat. # MUPAKT-TOT, # MPLM, # MUPA and # MATF, respectively).

Techniques: Two Tailed Test

Journal: Journal of Cell Science

Article Title: Urokinase-type plasminogen activator-mediated crosstalk between N-cadherin and β-catenin promotes wound healing

doi: 10.1242/jcs.255919

Figure Lengend Snippet: uPA inhibits the degradation of β-catenin. (A–F) Representative western blot analyses (A,C,E) and quantification of the intensity of the band (B,D,F) of total β-catenin (A–D), and active β-catenin (E,F) in Wt astrocytes incubated for 30 min (A,C), or for 0, 15 and 30 min (E) with vehicle (control, C) or 5 nM of either uPA (A,E) or its ATF (C). n=5 (B,D) and n=6 (F). Unpaired two-tailed Student's t-test was performed in B,D; one-way ANOVA with Dunnett's multiple comparisons test in F. (G) Mean growth of a monolayer of Wt astrocytes 24 h after a mechanical injury and treatment with vehicle (control; n=17), or 5 nM of uPA (n=25), or a combination of 5 nM of uPA and 1 μM of XAV-939 (n=24), or with XAV-939 alone (n=19). Results are presented as percentage of growth of wounded vehicle (control)-treated Wt astrocytes. Two-way ANOVA with Holm-Sidak's multiple comparisons test. In all box plots, the box represents the 25–75th percentiles, and the median is indicated. The whiskers show the range.

Article Snippet: The ELISA assay for uPA, and recombinant plasmin, murine uPA and its N-terminal fragment (ATF) were purchased from Molecular Innovations (Novi, MI; Cat. # MUPAKT-TOT, # MPLM, # MUPA and # MATF, respectively).

Techniques: Western Blot, Incubation, Two Tailed Test