chemerin neutralizing antibody  (Bio-Techne corporation)

Journal: PLoS ONE

Article Title: Elastase and Tryptase Govern TNFα-Mediated Production of Active Chemerin by Adipocytes

doi: 10.1371/journal.pone.0051072

Figure Lengend Snippet: Twenty-four hour conditioned media from 3T3-L1 ( A , C ) or BMSC ( B ) adipocytes treated with 20 ng mL −1 TNFα or an equivalent volume of the 0.1% BSA/PBS vehicle control were either incubated for 1 hour with 10 µg mL −1 of goat anti-mouse chemerin neutralization antibody or 10 µg mL −1 IgG control antibody ( A, B ), or separated by size using exclusion column with a 10 kDa molecular weight cutoff ( C ) prior to analysis by CMKLR1 bioassay. All bars represent the mean ± s.e.m. of 3 samples and are representative of 3 independent experiments. *P<0.05 compared to the goat IgG, 0.1% PBS/BSA control ( A, B ) or the 0.1% BSA/PBS control ( C ) and † P<0.05 compared to the within group goat IgG control ( A, B ) and the respective >10 kDa group ( C ), two-way ANOVA, followed by Bonferroni’s post hoc test.

Article Snippet: The specific activation of the CMKLR1 bioassay by chemerin was determined by incubating the 24 h conditioned media from 3T3-L1 or BMSC-derived adipocytes for 1 h with 10 μg mL −1 of goat anti-mouse chemerin neutralization antibody (R&D Systems) or 10 μg mL −1 of goat IgG control antibody (Life Technologies Inc.) prior to analyzing the samples in the CMKLR1 bioassay.

Techniques: Control, Incubation, Neutralization, Molecular Weight, Bioassay

Journal: PLoS ONE

Article Title: Elastase and Tryptase Govern TNFα-Mediated Production of Active Chemerin by Adipocytes

doi: 10.1371/journal.pone.0051072

Figure Lengend Snippet: 3T3-L1 adipocytes were treated for 24 hours with 20 ng mL −1 TNFα or 0.1% BSA/PBS vehicle control in combination with a 1∶200 dilution of a protease inhibitor cocktail (PIC) or its respective vehicle (1∶200 diluted DMSO) prior to analysis by CMKLR1 bioassay ( A, B ) or western blot ( C, D ). All bars represent the mean ± s.e.m. of 3 samples, and are representative of 3 independent experiments. Western blot analysis using an R&D anti-chemerin antibody is representative of 4 samples per group and 3 independent experiments. *P<0.05 compared to the 0.1% BSA/PBS, 1∶200 DMSO vehicle control, two-way ANOVA, followed by Bonferroni’s post hoc test.

Article Snippet: The specific activation of the CMKLR1 bioassay by chemerin was determined by incubating the 24 h conditioned media from 3T3-L1 or BMSC-derived adipocytes for 1 h with 10 μg mL −1 of goat anti-mouse chemerin neutralization antibody (R&D Systems) or 10 μg mL −1 of goat IgG control antibody (Life Technologies Inc.) prior to analyzing the samples in the CMKLR1 bioassay.

Techniques: Control, Protease Inhibitor, Bioassay, Western Blot

Journal: PLoS ONE

Article Title: Elastase and Tryptase Govern TNFα-Mediated Production of Active Chemerin by Adipocytes

doi: 10.1371/journal.pone.0051072

Figure Lengend Snippet: 3T3-L1 or BMSC adipocytes were treated with TNFα or 0.1% BSA/PBS vehicle control in combination with 0–30 µM of the serine protease inhibitor aprotinin or 0–100 µM of the cysteine protease inhibitor E-64 or an equivalent volume of their respective vehicle controls, water or 0.9% NaCl. The effect of these treatments on the apparent concentration of active chemerin in adipocyte media was measured by the CMKLR1 bioassay (A–C). The effect of these treatments on the immunodetectable levels of total chemerin in adipocyte media was measured by western blot and quantified by densitometry (D–F). For densitometry analysis, the dual vehicle treatment (i.e. 0.1% BSA/PBS with H 2 0 or 0.9% NaCl) served as the reference control and was assigned a value of 100%. All bars represent the mean ± s.e.m. of 3 samples and are representative of 3 independent experiments. Western blot using an R&D anti-chemerin antibody is representative of 4 samples per group and 3 independent experiments. * P<0.05 compared to the within group 0.1% BSA/PBS vehicle control, † P<0.05 compared to the TNFα/vehicle control groups, two-way ANOVA, followed by Bonferroni’s post hoc test (A–C).

Article Snippet: The specific activation of the CMKLR1 bioassay by chemerin was determined by incubating the 24 h conditioned media from 3T3-L1 or BMSC-derived adipocytes for 1 h with 10 μg mL −1 of goat anti-mouse chemerin neutralization antibody (R&D Systems) or 10 μg mL −1 of goat IgG control antibody (Life Technologies Inc.) prior to analyzing the samples in the CMKLR1 bioassay.

Techniques: Control, Protease Inhibitor, Concentration Assay, Bioassay, Western Blot

Journal: PLoS ONE

Article Title: Elastase and Tryptase Govern TNFα-Mediated Production of Active Chemerin by Adipocytes

doi: 10.1371/journal.pone.0051072

Figure Lengend Snippet: The concentrations of elastase, tryptase, and tPA in 24 hour conditioned media from 3T3-L1 and BMSC adipocytes treated with 20 ng mL −1 of TNFα or equivalent volume of 0.1% BSA/PBS vehicle control were measured by western blot analysis (A). The apparent concentration of active chemerin in 24 h conditioned media from 3T3-L1 adipocytes treated with 20 ng mL −1 of TNFα (B) or equivalent volume of 0.1% BSA/PBS (B, Inset) together with neutralizing antibodies for elastase and tryptase (alone or in combination) or IgG control was measured using the CMKLR1 bioassay. All bars represent the mean ± s.e.m. of 3 samples, and are representative of 2 independent experiments. Western blots are representative of 4 samples per group and 3 independent experiments. * P<0.05 compared to the TNFα/IgG or the 0.1% BSA/PBS/IgG (Inset) treated cells, † P<0.05 compared to the TNFα+anti-elastase or anti-tryptase treated cells, two-way ANOVA, followed by Bonferroni’s post hoc test.

Article Snippet: The specific activation of the CMKLR1 bioassay by chemerin was determined by incubating the 24 h conditioned media from 3T3-L1 or BMSC-derived adipocytes for 1 h with 10 μg mL −1 of goat anti-mouse chemerin neutralization antibody (R&D Systems) or 10 μg mL −1 of goat IgG control antibody (Life Technologies Inc.) prior to analyzing the samples in the CMKLR1 bioassay.

Techniques: Control, Western Blot, Concentration Assay, Bioassay

Journal: PLoS ONE

Article Title: Elastase and Tryptase Govern TNFα-Mediated Production of Active Chemerin by Adipocytes

doi: 10.1371/journal.pone.0051072

Figure Lengend Snippet: CMKLR1 bioassay and western blot analysis were used to identify the effect of bestatin, an inhibitor of aminopeptidases, alone and in combination with TNFα or a vehicle control on the apparent ( A ) and total ( B ) media chemerin concentration of 3T3-L1 adipocytes. All bars represent the mean ± s.e.m. of 3 samples and are representative of 3 independent experiments. Western blot using an R&D anti-chemerin antibody is representative of 4 samples per group and 3 independent experiments. † P<0.05 compared to the TNFα/vehicle control, two-way ANOVA followed by Bonferroni’s post hoc test ( A ).

Article Snippet: The specific activation of the CMKLR1 bioassay by chemerin was determined by incubating the 24 h conditioned media from 3T3-L1 or BMSC-derived adipocytes for 1 h with 10 μg mL −1 of goat anti-mouse chemerin neutralization antibody (R&D Systems) or 10 μg mL −1 of goat IgG control antibody (Life Technologies Inc.) prior to analyzing the samples in the CMKLR1 bioassay.

Techniques: Bioassay, Western Blot, Control, Concentration Assay

Journal: PLoS ONE

Article Title: Elastase and Tryptase Govern TNFα-Mediated Production of Active Chemerin by Adipocytes

doi: 10.1371/journal.pone.0051072

Figure Lengend Snippet: Our findings together support a model of adipocyte-derived proteolytic control of chemerin activity. Under basal conditions the activity of adipocyte-secreted chemerin (1) at CMKLR1 is determined by a precise balance between activation by serine and cysteine protease (2) and deactivation by aminopeptidases (3). Following treatment with TNFα, elevated secretion of chemerin (4) and production of elastase and tryptase (5) alter this balance resulting in increased concentration of a chemerin product(s) with high activity towards CMKLR1 (6).

Article Snippet: The specific activation of the CMKLR1 bioassay by chemerin was determined by incubating the 24 h conditioned media from 3T3-L1 or BMSC-derived adipocytes for 1 h with 10 μg mL −1 of goat anti-mouse chemerin neutralization antibody (R&D Systems) or 10 μg mL −1 of goat IgG control antibody (Life Technologies Inc.) prior to analyzing the samples in the CMKLR1 bioassay.

Techniques: Derivative Assay, Control, Activity Assay, Activation Assay, Concentration Assay

Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: Epithelial cells in fetal intestine produce chemerin to recruit macrophages

doi: 10.1152/ajpgi.90730.2008

Figure Lengend Snippet: Fetal IECS secrete chemerin. Western blot shows chemerin detected in culture media conditioned with primary IECs from fetal intestinal tissue. Chemerin concentrations increased with gestation in E-CMs prepared from fetal intestinal tissue from 10–14, 15–19, and 20–24 wk. Chemerin was immunoprecipitated from E-CMs by using a polyclonal goat anti-chemerin antibody and immobilized protein A/G, resolved in 15% polyacrylamide gels, and detected by use of a murine monoclonal anti-chemerin antibody. The blot shows E-CMs prepared from 3 different fetuses in each group. Bar diagram (means ± SE) shows average densitometric analysis of the blots. Groups were compared by the Kruskal-Wallis H-test/Dunn's multiple-comparison posttest.

Article Snippet: In some wells, we added the E-CMs after preincubation for 30 min with excess neutralizing anti-chemerin antibody (10 μg/ml; R&D).

Techniques: Western Blot, Immunoprecipitation

Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: Epithelial cells in fetal intestine produce chemerin to recruit macrophages

doi: 10.1152/ajpgi.90730.2008

Figure Lengend Snippet: Macrophage chemoattractant activity of 10- to 24-wk fetal epithelial-conditioned media correlates with chemerin concentrations. A: the number of intestinal macrophages increases with gestation during the 10- to 24-wk period, in correlation with increasing epithelial expression of chemerin. Photomicrographs (×1,000) showing the HAM56 (a pan-macrophage marker) immunoreactivity in macrophages (red). Nuclear staining was obtained with DAPI (blue). The number of macrophages in the lamina propria was normalized against the number of epithelial nuclei at that level. Bar diagram (means ± SE) shows that the number of macrophages in the lamina propria increases with gestation in the 10- to 24-wk period, which also correlates with increasing chemerin expression (Figs. 1 and ​and2).2). Data are representative of 3 different fetuses in each group. Macrophages were enumerated in 10 different villi in each section. B: intestinal macrophages express CMKLR1. Photomicrographs (×1,000) showing the colocalization of HAM56 (red) and CMKLR1 (the cognate receptor for chemerin; green) in intestinal tissue from a 22-wk fetus. The colocalization is seen as yellow in a computer-merged image. Nuclear staining was obtained with DAPI (blue). Data are representative of sections from 3 different fetuses. C: macrophage chemoattractant activity of 10- to 24-wk fetal epithelial-conditioned media correlates with chemerin concentrations. The macrophage chemoattractant activity of fetal E-CMs increased with gestational age, in correlation with increasing concentrations of chemerin in these E-CMs (Fig. 2). The chemotactic activity of fetal E-CMs on monocyte-derived macrophages was measured in a fluorescence-based assay. Cord blood monocytes were differentiated in vitro for 5 days and these monocyte-derived macrophages were stained green with calcein-AM (a colorless dye that is cleaved by intracellular esterases to release a green fluorescent molecule). Bar diagram shows the mean number (± SE) of macrophages migrating to standards or test samples. Standards (shaded bars) included a range of concentrations of recombinant chemerin (10–100 pM). The chemotactic activity was inhibited in the presence of excess neutralizing anti-chemerin antibody (hatched bar). Groups were compared by Kruskal-Wallis H-test/Dunn's multiple-comparison posttest. Data represent 3 separate experiments. These studies included monocyte-derived macrophages derived from adult peripheral blood as positive control for chemerin-induced macrophage chemotaxis (not depicted). Inset a: FACS profile showing the expression of CMKLR1 on macrophages. Inset b: photomicrograph showing calcein-stained macrophages (magnification ×100).

Article Snippet: In some wells, we added the E-CMs after preincubation for 30 min with excess neutralizing anti-chemerin antibody (10 μg/ml; R&D).

Techniques: Activity Assay, Expressing, Marker, Staining, Derivative Assay, Fluorescence, In Vitro, Recombinant, Positive Control, Chemotaxis Assay