recombinant human ccl16  (OriGene)

Journal: International journal of molecular sciences

Article Title: Distinct Plasma Immune Profile in ALS Implicates sTNFR-II in pAMPK/Leptin Homeostasis.

doi: 10.3390/ijms24065065

Figure Lengend Snippet: Figure 3. Plasma levels of leptin, CCL16 and sTNF-RII measured by ELISA. (a) concentration (ng/mL) of leptin measured by ELISA in healthy controls (38) and sALS (51). (b,d) Concentration (ng/mL) of leptin (b), CCL16 (c) and sTNF-RII (d) measured by ELISA in slow (40) and fast (11) progressing ALS patients. (e,f) Concentration (ng/mL) of leptin in sALS and controls sub-grouped by sex (e) and by sex and rate of progression (f) (Data are mean ± SEM * p ≤0.05, ** p < 0.01, **** p < 0.0001, NS, not significant).

Article Snippet: Cells were also treated with different concentrations of recombinant CCL16 (R&D systems, Minneapolis, MN, USA, # 802-HC-025) or recombinant sTNF-RII (MyBioSource, MBS343136, London, UK).

Techniques: Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: Frontiers in Immunology

Article Title: Characterizing ligand-receptor interactions and unveiling the pro-tumorigenic role of CCL16-CCR1 axis in the microenvironment of hepatocellular carcinoma

doi: 10.3389/fimmu.2023.1299953

Figure Lengend Snippet: Reagent information used in this study.

Article Snippet: Human CCL16 ELISA kit , Abcam , Ab243673.

Techniques: Enzyme-linked Immunosorbent Assay, Magnetic Beads, Transfection, Recombinant

Journal: Frontiers in Immunology

Article Title: Characterizing ligand-receptor interactions and unveiling the pro-tumorigenic role of CCL16-CCR1 axis in the microenvironment of hepatocellular carcinoma

doi: 10.3389/fimmu.2023.1299953

Figure Lengend Snippet: Primer sequences for qPCR.

Article Snippet: Human CCL16 ELISA kit , Abcam , Ab243673.

Techniques:

Journal: Frontiers in Immunology

Article Title: Characterizing ligand-receptor interactions and unveiling the pro-tumorigenic role of CCL16-CCR1 axis in the microenvironment of hepatocellular carcinoma

doi: 10.3389/fimmu.2023.1299953

Figure Lengend Snippet: Tumor cell-derived CCL16 mediates the recruitment and M2 polarization of macrophages in the liver cancer microenvironment. (A) Expression of CCL16 and CCR1 in different cell types at the single-cell level. (B) Expression levels of CCL16 in different liver cancer cell lines from the CCLE database. (C) mRNA expression of CCL16 in different liver cancer cell lines detected by qPCR. (D) Validation of CCL16 knockdown in HEPG2 cell line. (E) Validation of CCL16 overexpression in SNU761 cell line. (F) Transwell assay to evaluate the recruitment of THP1 cells by control and CCL16 knockdown HEPG2 cells, and ELISA assay to measure CCL16 concentration in the culture supernatant of HEPG2 cells. (G) Transwell assay to evaluate the recruitment of THP1 cells by control and CCL16 overexpressing SNU761 cells, and ELISA assay to measure CCL16 concentration in the culture supernatant of SNU761 cells. (H) Schematic diagram illustrating the cell co-culture system and the macrophage migration assay. (I) Flow cytometry analysis to detect the proportion of M2-polarized cells after co-culture of THP1 cells with CCL16 knockdown or overexpressing tumor cells. Transwell Scale Bar = 100μm. Three independent replicates were conducted. Statistical data are presented as mean ± SD, and each data point represents an independent measurement. Unpaired Student’s t-test. ns, not significant; **: P < 0.01; ***: P < 0.001; ****: P < 0.0001.

Article Snippet: Human CCL16 ELISA kit , Abcam , Ab243673.

Techniques: Derivative Assay, Expressing, Over Expression, Transwell Assay, Enzyme-linked Immunosorbent Assay, Concentration Assay, Co-Culture Assay, Migration, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: Characterizing ligand-receptor interactions and unveiling the pro-tumorigenic role of CCL16-CCR1 axis in the microenvironment of hepatocellular carcinoma

doi: 10.3389/fimmu.2023.1299953

Figure Lengend Snippet: The recruitment of tumor-associated macrophages mediated by CCL16 depends on the macrophage receptor CCR1. (A) Immunoprecipitation assay to detect the interaction between Flag-CCL16 and CCR1, CCR2, CCR5, CCR8 in THP1 cell culture medium. (B) Immunofluorescence assay to detect the co-localization of Flag-CCL16 and CCR1 in THP1 cells after the addition of Flag-CCL16. Scale bar = 20 μm. (C) qPCR validation of CCR1 knockdown in THP1 cells. (D) Transwell assay to evaluate the cell migration ability of CCR1 knockdown THP1 cells co-cultured with HEPG2 cells. (E) Recruitment of THP1 cells by control or 5μM BX471-treated CCL16 overexpressing SNU761 cells after 24 hours. (F) Recruitment of THP1 cells by CCL16 overexpressing SNU761 cells after CCR1 knockdown. Transwell Scale Bar = 100μm. Three independent replicates were conducted. Statistical data are presented as mean ± SD, and each data point represents an independent measurement. Unpaired Student’s t-test or two-way ANOVA was used for statistical analysis. ns, not significant; ***: P < 0.001; ****: P < 0.0001.

Article Snippet: Human CCL16 ELISA kit , Abcam , Ab243673.

Techniques: Immunoprecipitation, Cell Culture, Immunofluorescence, Transwell Assay, Migration

Journal: Frontiers in Immunology

Article Title: Characterizing ligand-receptor interactions and unveiling the pro-tumorigenic role of CCL16-CCR1 axis in the microenvironment of hepatocellular carcinoma

doi: 10.3389/fimmu.2023.1299953

Figure Lengend Snippet: Clinical characteristics of patients with high and low expression of CCL16.

Article Snippet: Human CCL16 ELISA kit , Abcam , Ab243673.

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: Characterizing ligand-receptor interactions and unveiling the pro-tumorigenic role of CCL16-CCR1 axis in the microenvironment of hepatocellular carcinoma

doi: 10.3389/fimmu.2023.1299953

Figure Lengend Snippet: High expression of CCL16 is positively correlated with the infiltration of M2 macrophages and the expression of CCR1 in clinical tissues. (A) Immunohistochemistry representative images and Pearson correlation analysis of CCL16 with CCR1, CD68, and CD206 in liver cancer patient samples, as well as the Mean Optical Density (MOD) values. Scale Bar = 100μm. (B) Immunofluorescence detection of CCR1+ macrophage infiltration. Scale Bar = 20μm. Statistical analysis of the difference in CD68+CCR1+ cell numbers between high and low CCL16 expression groups using unpaired Student’s t-test. **: P < 0.01. Pearson correlation analysis of CCL16 expression and CCR1+ macrophage infiltration, r = 0.5743, P < 0.001.

Article Snippet: Human CCL16 ELISA kit , Abcam , Ab243673.

Techniques: Expressing, Immunohistochemistry, Immunofluorescence

Journal: Frontiers in Immunology

Article Title: Characterizing ligand-receptor interactions and unveiling the pro-tumorigenic role of CCL16-CCR1 axis in the microenvironment of hepatocellular carcinoma

doi: 10.3389/fimmu.2023.1299953

Figure Lengend Snippet: Reagent information used in this study.

Article Snippet: CCL16 expression in the control, CCL16-knockdown HEPG2 cells, and CCL16-overexpressing SNU761 cells were measured using human CCL16 ELISA kits (Abcam).

Techniques: Enzyme-linked Immunosorbent Assay, Magnetic Beads, Transfection, Recombinant

Journal: Frontiers in Immunology

Article Title: Characterizing ligand-receptor interactions and unveiling the pro-tumorigenic role of CCL16-CCR1 axis in the microenvironment of hepatocellular carcinoma

doi: 10.3389/fimmu.2023.1299953

Figure Lengend Snippet: Primer sequences for qPCR.

Article Snippet: CCL16 expression in the control, CCL16-knockdown HEPG2 cells, and CCL16-overexpressing SNU761 cells were measured using human CCL16 ELISA kits (Abcam).

Techniques:

Journal: Frontiers in Immunology

Article Title: Characterizing ligand-receptor interactions and unveiling the pro-tumorigenic role of CCL16-CCR1 axis in the microenvironment of hepatocellular carcinoma

doi: 10.3389/fimmu.2023.1299953

Figure Lengend Snippet: Tumor cell-derived CCL16 mediates the recruitment and M2 polarization of macrophages in the liver cancer microenvironment. (A) Expression of CCL16 and CCR1 in different cell types at the single-cell level. (B) Expression levels of CCL16 in different liver cancer cell lines from the CCLE database. (C) mRNA expression of CCL16 in different liver cancer cell lines detected by qPCR. (D) Validation of CCL16 knockdown in HEPG2 cell line. (E) Validation of CCL16 overexpression in SNU761 cell line. (F) Transwell assay to evaluate the recruitment of THP1 cells by control and CCL16 knockdown HEPG2 cells, and ELISA assay to measure CCL16 concentration in the culture supernatant of HEPG2 cells. (G) Transwell assay to evaluate the recruitment of THP1 cells by control and CCL16 overexpressing SNU761 cells, and ELISA assay to measure CCL16 concentration in the culture supernatant of SNU761 cells. (H) Schematic diagram illustrating the cell co-culture system and the macrophage migration assay. (I) Flow cytometry analysis to detect the proportion of M2-polarized cells after co-culture of THP1 cells with CCL16 knockdown or overexpressing tumor cells. Transwell Scale Bar = 100μm. Three independent replicates were conducted. Statistical data are presented as mean ± SD, and each data point represents an independent measurement. Unpaired Student’s t-test. ns, not significant; **: P < 0.01; ***: P < 0.001; ****: P < 0.0001.

Article Snippet: CCL16 expression in the control, CCL16-knockdown HEPG2 cells, and CCL16-overexpressing SNU761 cells were measured using human CCL16 ELISA kits (Abcam).

Techniques: Derivative Assay, Expressing, Over Expression, Transwell Assay, Enzyme-linked Immunosorbent Assay, Concentration Assay, Co-Culture Assay, Migration, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: Characterizing ligand-receptor interactions and unveiling the pro-tumorigenic role of CCL16-CCR1 axis in the microenvironment of hepatocellular carcinoma

doi: 10.3389/fimmu.2023.1299953

Figure Lengend Snippet: The recruitment of tumor-associated macrophages mediated by CCL16 depends on the macrophage receptor CCR1. (A) Immunoprecipitation assay to detect the interaction between Flag-CCL16 and CCR1, CCR2, CCR5, CCR8 in THP1 cell culture medium. (B) Immunofluorescence assay to detect the co-localization of Flag-CCL16 and CCR1 in THP1 cells after the addition of Flag-CCL16. Scale bar = 20 μm. (C) qPCR validation of CCR1 knockdown in THP1 cells. (D) Transwell assay to evaluate the cell migration ability of CCR1 knockdown THP1 cells co-cultured with HEPG2 cells. (E) Recruitment of THP1 cells by control or 5μM BX471-treated CCL16 overexpressing SNU761 cells after 24 hours. (F) Recruitment of THP1 cells by CCL16 overexpressing SNU761 cells after CCR1 knockdown. Transwell Scale Bar = 100μm. Three independent replicates were conducted. Statistical data are presented as mean ± SD, and each data point represents an independent measurement. Unpaired Student’s t-test or two-way ANOVA was used for statistical analysis. ns, not significant; ***: P < 0.001; ****: P < 0.0001.

Article Snippet: CCL16 expression in the control, CCL16-knockdown HEPG2 cells, and CCL16-overexpressing SNU761 cells were measured using human CCL16 ELISA kits (Abcam).

Techniques: Immunoprecipitation, Cell Culture, Immunofluorescence, Transwell Assay, Migration

Journal: Frontiers in Immunology

Article Title: Characterizing ligand-receptor interactions and unveiling the pro-tumorigenic role of CCL16-CCR1 axis in the microenvironment of hepatocellular carcinoma

doi: 10.3389/fimmu.2023.1299953

Figure Lengend Snippet: Clinical characteristics of patients with high and low expression of CCL16.

Article Snippet: CCL16 expression in the control, CCL16-knockdown HEPG2 cells, and CCL16-overexpressing SNU761 cells were measured using human CCL16 ELISA kits (Abcam).

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: Characterizing ligand-receptor interactions and unveiling the pro-tumorigenic role of CCL16-CCR1 axis in the microenvironment of hepatocellular carcinoma

doi: 10.3389/fimmu.2023.1299953

Figure Lengend Snippet: High expression of CCL16 is positively correlated with the infiltration of M2 macrophages and the expression of CCR1 in clinical tissues. (A) Immunohistochemistry representative images and Pearson correlation analysis of CCL16 with CCR1, CD68, and CD206 in liver cancer patient samples, as well as the Mean Optical Density (MOD) values. Scale Bar = 100μm. (B) Immunofluorescence detection of CCR1+ macrophage infiltration. Scale Bar = 20μm. Statistical analysis of the difference in CD68+CCR1+ cell numbers between high and low CCL16 expression groups using unpaired Student’s t-test. **: P < 0.01. Pearson correlation analysis of CCL16 expression and CCR1+ macrophage infiltration, r = 0.5743, P < 0.001.

Article Snippet: CCL16 expression in the control, CCL16-knockdown HEPG2 cells, and CCL16-overexpressing SNU761 cells were measured using human CCL16 ELISA kits (Abcam).

Techniques: Expressing, Immunohistochemistry, Immunofluorescence

Journal: Biomolecules

Article Title: Structure and Dynamics of Human Chemokine CCL16—Implications for Biological Activity

doi: 10.3390/biom12111588

Figure Lengend Snippet: Data collection and refinement statistics (r.m.s., root-mean-square). Values in parentheses refer to the highest-resolution shell.

Article Snippet: A codon-optimised synthetic cDNA coding for mature human CCL16 was obtained from Life Technologies (Darmstadt, Germany) and ligated into a modified pET-15b vector using PspOMI and XhoI restriction sites.

Techniques:

Journal: Biomolecules

Article Title: Structure and Dynamics of Human Chemokine CCL16—Implications for Biological Activity

doi: 10.3390/biom12111588

Figure Lengend Snippet: CCL16 (C-C motif ligand 16) is preferentially expressed in the liver. ( A ) The CCL16 expression of normal human primary keratinocytes, dermal fibroblasts, microvascular endothelial cells, and hepatocytes ( n = 5 each), given as relative units (RU) normalised to 18S. ( B ) The CCL16 expression profile in the BIGE (Body Index of human Gene Expression) database representing 104 different cell types and tissues. ( C ) The immunohistochemistry of CCL16 protein expression in frozen liver sections (original magnification: ×100).

Article Snippet: A codon-optimised synthetic cDNA coding for mature human CCL16 was obtained from Life Technologies (Darmstadt, Germany) and ligated into a modified pET-15b vector using PspOMI and XhoI restriction sites.

Techniques: Expressing, Immunohistochemistry

Journal: Biomolecules

Article Title: Structure and Dynamics of Human Chemokine CCL16—Implications for Biological Activity

doi: 10.3390/biom12111588

Figure Lengend Snippet: CCL16 expression in the liver occurs homeostatically. CCL16 mRNA was quantified in normal human liver tissue ( n = 4) in comparison to livers with cirrhosis ( n = 7), hepatitis B virus (HBV) and hepatitis C virus (HCV) infection ( n = 4–5), as well as cholangiocarcinoma (CCC; n = 7) and hepatocellular carcinoma (HCC; n = 7). Values are expressed as relative units (RU) normalised to 18S. Measurements of individual samples and the mean of each group are shown. A Mann–Whitney U test was performed to assess statistical significance (* p < 0.05).

Article Snippet: A codon-optimised synthetic cDNA coding for mature human CCL16 was obtained from Life Technologies (Darmstadt, Germany) and ligated into a modified pET-15b vector using PspOMI and XhoI restriction sites.

Techniques: Expressing, Infection, MANN-WHITNEY

Journal: Biomolecules

Article Title: Structure and Dynamics of Human Chemokine CCL16—Implications for Biological Activity

doi: 10.3390/biom12111588

Figure Lengend Snippet: The liver is the primary source of CCL16 in human serum. CCL16 protein abundance was measured in the sera of patients who underwent liver resection. One group represents a removal of more than 30% of the liver mass, the other group had less than 30% removed. Sera were taken at the end of surgery (0 h) and at various time points thereafter and analysed by ELISA. Data are represented by individual values as well as means for each time point. A Mann–Whitney U test was performed to analyse statistical differences (* p < 0.05; ** p < 0.01).

Article Snippet: A codon-optimised synthetic cDNA coding for mature human CCL16 was obtained from Life Technologies (Darmstadt, Germany) and ligated into a modified pET-15b vector using PspOMI and XhoI restriction sites.

Techniques: Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: Biomolecules

Article Title: Structure and Dynamics of Human Chemokine CCL16—Implications for Biological Activity

doi: 10.3390/biom12111588

Figure Lengend Snippet: The crystal structure of human CCL16. ( A ) CCL16 forms a canonical CC-type dimer. One of the protomers (chain B, right) is shown in ribbon representation, the other (chain A, left) as a Cα trace, on which the prototypical family member CCL5 (PDB-ID 1U4L , chain A, yellow) is superimposed for comparison. The C-terminal extension relative to CCL5 is coloured dark grey. ( B ) Close-up view of chain B, corresponding to the boxed area in ( A ), with a focus on the C-terminal extension and its interaction with the chemokine core domain. In the segments shown in all-atom mode, most side chains have been truncated for visual clarity. Electron density (2 mF o - DF c synthesis, contoured at 1.2 σ) is displayed for residues 67–75; hydrogen bonds stabilising the conformation of the terminus are indicated by dotted lines. ( C ) Sequence alignment of CCL16 with other human CC-type chemokines, including CCL23 and CCL24, which carry unique extensions at their N- and C-termini, respectively. The degree of sequence conservation is indicated by shading; green diamonds mark the invariant cysteine residues, and purple triangles identify the BBxB motif in the β2-β3 loop and an additional basic residue in the N-loop, both of which are implicated in GAG binding. The secondary structure of CCL16 (chain B of our crystal structure) is depicted above the alignment.

Article Snippet: A codon-optimised synthetic cDNA coding for mature human CCL16 was obtained from Life Technologies (Darmstadt, Germany) and ligated into a modified pET-15b vector using PspOMI and XhoI restriction sites.

Techniques: Sequencing, Binding Assay

Journal: Biomolecules

Article Title: Structure and Dynamics of Human Chemokine CCL16—Implications for Biological Activity

doi: 10.3390/biom12111588

Figure Lengend Snippet: Potential GAG interaction modes of CCL16. The crystal structure of CCL16 (surface representation, oriented as in A) is shown in the context of two published models featuring short mucopolysaccharide chains docked to CCL5. The chondroitin sulfate (CS, panel A ) and heparin (Hep, panel B ) moieties are drawn in space-filling mode; black patches on the CCL16 surface indicate basic residues K47, R48, and R50 of the BBxB motif (#) as well as K21 (*), suggested to mediate this interaction by analogy to CCL5. The electrostatic potential of CCL16 is contoured at +3 kT/e (blue) and −3 kT/e (red).

Article Snippet: A codon-optimised synthetic cDNA coding for mature human CCL16 was obtained from Life Technologies (Darmstadt, Germany) and ligated into a modified pET-15b vector using PspOMI and XhoI restriction sites.

Techniques:

Journal: Biomolecules

Article Title: Structure and Dynamics of Human Chemokine CCL16—Implications for Biological Activity

doi: 10.3390/biom12111588

Figure Lengend Snippet: The free energy surface of CCL16. The free energy ΔG (given in kJ mol −1 relative to the lowest energy state) is plotted against the two main motion vectors representing the C-terminal flexibility. Structures 1–4 correspond to the free energy minima labelled in the diagram. Conformer 1 represents the global minimum, i.e., ΔG = 0 kJ mol −1 . Conformers 1 and 2 have their C-termini positioned laterally to the protein core, interacting with the GAG binding domain of the opposite chain. In conformers 3 and 4, the C-termini interact with each other above the groove formed between the two CCL16 chains. Residues Q1–L73 are shown as a molecular surface with the GAG binding segments (K47–R50) highlighted in yellow. C-termini (P74–Q97) are rendered as ribbons, where chains A and B are coloured blue and red, respectively. For further structural details consult .

Article Snippet: A codon-optimised synthetic cDNA coding for mature human CCL16 was obtained from Life Technologies (Darmstadt, Germany) and ligated into a modified pET-15b vector using PspOMI and XhoI restriction sites.

Techniques: Binding Assay

Journal: Biomolecules

Article Title: Structure and Dynamics of Human Chemokine CCL16—Implications for Biological Activity

doi: 10.3390/biom12111588

Figure Lengend Snippet: Enrichment of CCL16 from human serum. Heparin-binding proteins were enriched from the sera of human subjects ( n = 3) via heparin affinity chromatography. Expression was determined by immunoblotting analysis using a polyclonal goat anti-human CCL16 antibody. As a control, the fourth lane contains 0.4 ng of recombinant human (rh) CCL16. The identity of the immunoreactive band with about 55 kDa apparent mass is unknown.

Article Snippet: A codon-optimised synthetic cDNA coding for mature human CCL16 was obtained from Life Technologies (Darmstadt, Germany) and ligated into a modified pET-15b vector using PspOMI and XhoI restriction sites.

Techniques: Binding Assay, Affinity Chromatography, Expressing, Western Blot, Recombinant

Journal: Biomolecules

Article Title: Structure and Dynamics of Human Chemokine CCL16—Implications for Biological Activity

doi: 10.3390/biom12111588

Figure Lengend Snippet: Data collection and refinement statistics (r.m.s., root-mean-square). Values in parentheses refer to the highest-resolution shell.

Article Snippet: Sections were stained with mouse anti-human CCL16 monoclonal IgG1 antibody (R&D Systems, Wiesbaden, Germany), using appropriate isotype antibodies as negative controls.

Techniques:

Journal: Biomolecules

Article Title: Structure and Dynamics of Human Chemokine CCL16—Implications for Biological Activity

doi: 10.3390/biom12111588

Figure Lengend Snippet: CCL16 (C-C motif ligand 16) is preferentially expressed in the liver. ( A ) The CCL16 expression of normal human primary keratinocytes, dermal fibroblasts, microvascular endothelial cells, and hepatocytes ( n = 5 each), given as relative units (RU) normalised to 18S. ( B ) The CCL16 expression profile in the BIGE (Body Index of human Gene Expression) database representing 104 different cell types and tissues. ( C ) The immunohistochemistry of CCL16 protein expression in frozen liver sections (original magnification: ×100).

Article Snippet: Sections were stained with mouse anti-human CCL16 monoclonal IgG1 antibody (R&D Systems, Wiesbaden, Germany), using appropriate isotype antibodies as negative controls.

Techniques: Expressing, Gene Expression, Immunohistochemistry

Journal: Biomolecules

Article Title: Structure and Dynamics of Human Chemokine CCL16—Implications for Biological Activity

doi: 10.3390/biom12111588

Figure Lengend Snippet: CCL16 expression in the liver occurs homeostatically. CCL16 mRNA was quantified in normal human liver tissue ( n = 4) in comparison to livers with cirrhosis ( n = 7), hepatitis B virus (HBV) and hepatitis C virus (HCV) infection ( n = 4–5), as well as cholangiocarcinoma (CCC; n = 7) and hepatocellular carcinoma (HCC; n = 7). Values are expressed as relative units (RU) normalised to 18S. Measurements of individual samples and the mean of each group are shown. A Mann–Whitney U test was performed to assess statistical significance (* p < 0.05).

Article Snippet: Sections were stained with mouse anti-human CCL16 monoclonal IgG1 antibody (R&D Systems, Wiesbaden, Germany), using appropriate isotype antibodies as negative controls.

Techniques: Expressing, Comparison, Virus, Infection, MANN-WHITNEY

Journal: Biomolecules

Article Title: Structure and Dynamics of Human Chemokine CCL16—Implications for Biological Activity

doi: 10.3390/biom12111588

Figure Lengend Snippet: The liver is the primary source of CCL16 in human serum. CCL16 protein abundance was measured in the sera of patients who underwent liver resection. One group represents a removal of more than 30% of the liver mass, the other group had less than 30% removed. Sera were taken at the end of surgery (0 h) and at various time points thereafter and analysed by ELISA. Data are represented by individual values as well as means for each time point. A Mann–Whitney U test was performed to analyse statistical differences (* p < 0.05; ** p < 0.01).

Article Snippet: Sections were stained with mouse anti-human CCL16 monoclonal IgG1 antibody (R&D Systems, Wiesbaden, Germany), using appropriate isotype antibodies as negative controls.

Techniques: Quantitative Proteomics, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: Biomolecules

Article Title: Structure and Dynamics of Human Chemokine CCL16—Implications for Biological Activity

doi: 10.3390/biom12111588

Figure Lengend Snippet: The crystal structure of human CCL16. ( A ) CCL16 forms a canonical CC-type dimer. One of the protomers (chain B, right) is shown in ribbon representation, the other (chain A, left) as a Cα trace, on which the prototypical family member CCL5 (PDB-ID 1U4L , chain A, yellow) is superimposed for comparison. The C-terminal extension relative to CCL5 is coloured dark grey. ( B ) Close-up view of chain B, corresponding to the boxed area in ( A ), with a focus on the C-terminal extension and its interaction with the chemokine core domain. In the segments shown in all-atom mode, most side chains have been truncated for visual clarity. Electron density (2 mF o - DF c synthesis, contoured at 1.2 σ) is displayed for residues 67–75; hydrogen bonds stabilising the conformation of the terminus are indicated by dotted lines. ( C ) Sequence alignment of CCL16 with other human CC-type chemokines, including CCL23 and CCL24, which carry unique extensions at their N- and C-termini, respectively. The degree of sequence conservation is indicated by shading; green diamonds mark the invariant cysteine residues, and purple triangles identify the BBxB motif in the β2-β3 loop and an additional basic residue in the N-loop, both of which are implicated in GAG binding. The secondary structure of CCL16 (chain B of our crystal structure) is depicted above the alignment.

Article Snippet: Sections were stained with mouse anti-human CCL16 monoclonal IgG1 antibody (R&D Systems, Wiesbaden, Germany), using appropriate isotype antibodies as negative controls.

Techniques: Comparison, Sequencing, Residue, Binding Assay

Journal: Biomolecules

Article Title: Structure and Dynamics of Human Chemokine CCL16—Implications for Biological Activity

doi: 10.3390/biom12111588

Figure Lengend Snippet: Potential GAG interaction modes of CCL16. The crystal structure of CCL16 (surface representation, oriented as in A) is shown in the context of two published models featuring short mucopolysaccharide chains docked to CCL5. The chondroitin sulfate (CS, panel A ) and heparin (Hep, panel B ) moieties are drawn in space-filling mode; black patches on the CCL16 surface indicate basic residues K47, R48, and R50 of the BBxB motif (#) as well as K21 (*), suggested to mediate this interaction by analogy to CCL5. The electrostatic potential of CCL16 is contoured at +3 kT/e (blue) and −3 kT/e (red).

Article Snippet: Sections were stained with mouse anti-human CCL16 monoclonal IgG1 antibody (R&D Systems, Wiesbaden, Germany), using appropriate isotype antibodies as negative controls.

Techniques:

Journal: Biomolecules

Article Title: Structure and Dynamics of Human Chemokine CCL16—Implications for Biological Activity

doi: 10.3390/biom12111588

Figure Lengend Snippet: The free energy surface of CCL16. The free energy ΔG (given in kJ mol −1 relative to the lowest energy state) is plotted against the two main motion vectors representing the C-terminal flexibility. Structures 1–4 correspond to the free energy minima labelled in the diagram. Conformer 1 represents the global minimum, i.e., ΔG = 0 kJ mol −1 . Conformers 1 and 2 have their C-termini positioned laterally to the protein core, interacting with the GAG binding domain of the opposite chain. In conformers 3 and 4, the C-termini interact with each other above the groove formed between the two CCL16 chains. Residues Q1–L73 are shown as a molecular surface with the GAG binding segments (K47–R50) highlighted in yellow. C-termini (P74–Q97) are rendered as ribbons, where chains A and B are coloured blue and red, respectively. For further structural details consult .

Article Snippet: Sections were stained with mouse anti-human CCL16 monoclonal IgG1 antibody (R&D Systems, Wiesbaden, Germany), using appropriate isotype antibodies as negative controls.

Techniques: Binding Assay

Journal: Biomolecules

Article Title: Structure and Dynamics of Human Chemokine CCL16—Implications for Biological Activity

doi: 10.3390/biom12111588

Figure Lengend Snippet: Enrichment of CCL16 from human serum. Heparin-binding proteins were enriched from the sera of human subjects ( n = 3) via heparin affinity chromatography. Expression was determined by immunoblotting analysis using a polyclonal goat anti-human CCL16 antibody. As a control, the fourth lane contains 0.4 ng of recombinant human (rh) CCL16. The identity of the immunoreactive band with about 55 kDa apparent mass is unknown.

Article Snippet: Sections were stained with mouse anti-human CCL16 monoclonal IgG1 antibody (R&D Systems, Wiesbaden, Germany), using appropriate isotype antibodies as negative controls.

Techniques: Binding Assay, Affinity Chromatography, Expressing, Western Blot, Control, Recombinant

Journal: Journal of Animal Science and Biotechnology

Article Title: Gene expression of CCL8 and CXCL10 in peripheral blood leukocytes during early pregnancy in cows

doi: 10.1186/s40104-018-0263-z

Figure Lengend Snippet: Effects of interferon-τ (IFNT; 100 ng/mL) and CCL16 (100 ng/mL) on the mRNA expression of ( a ) CCL8 , ( b ) CXCL10 , ( c ) ISG15 , ( d ) MX1 and ( e ) MX2 in cultured monocytes, granulocytes and lymphocytes. Data are shown as percentage of the control value ( n = 5). Different letters indicate significant differences ( P < 0.05)

Article Snippet: Cultured leukocytes were further incubated in this medium with recombinant proteins as follows: bovine IFNT (100 ng/mL: 1.1 × 10 5 units/mg, generated from HEK293 cells as described previously [ ]) or recombinant human CCL16 (100 ng/mL: #TP723266, OriGene Technologies, Inc., Rockville, MD, USA).

Techniques: Expressing, Cell Culture, Control

Journal: International Journal of Molecular Sciences

Article Title: Possible Roles of CC- and CXC-Chemokines in Regulating Bovine Endometrial Function during Early Pregnancy

doi: 10.3390/ijms18040742

Figure Lengend Snippet: Comparison of mRNA levels for selected chemokines in the endometrium of pregnant vs. non-pregnant cows as determined by microarray analysis (Fold > 2.0; p < 0.05).

Article Snippet: Cultured endometrial tissues were further incubated in the medium with recombinant proteins as follows: bovine CCL2 (RP0027B, Kingfisher Biotech., Inc. St. Paul, MN, USA), human CCL8 (281-CP, R&D Systems, Inc. Minneapolis, MN, USA), bovine CCL11 (RP0071B, Kingfisher Biotech.), human CCL14 (1578-HC, R&D Systems), human CCL16 (TP723266, OriGene Technologies, Inc., Rockville, MD, USA), bovine CXCL10 (RP0079B, Kingfisher Biotech.), bovine tumor necrosis factor-α (TNF; 2279-BT, R&D Systems), human leukemia inhibitory factor (LIF; TP723270, OriGene Technologies), bovine IFNT (1.1 × 10 5 U/mg, generated from HEK293 cells as described previously; Takahashi et al., 2017 [ ]) or supernatant derived from homogenized fetal trophoblast on day 18 of pregnancy (FMP).

Techniques: Microarray

Journal: International Journal of Molecular Sciences

Article Title: Possible Roles of CC- and CXC-Chemokines in Regulating Bovine Endometrial Function during Early Pregnancy

doi: 10.3390/ijms18040742

Figure Lengend Snippet: Changes in relative amounts of mRNA for ( a ) CCL2, ( b ) CCL8, ( c ) CCL11, ( d ) CCL14, ( e ) CCL16, and ( f ) CXCL10 in the endometrium at days 15 and 18 of non-pregnant cows (NP) and pregnant cows (P). Data are means ± SEM of four cows per stage and are expressed as relative ratios of the mRNAs to SUZ12 polycomb repressive complex 2 subunit (SUZ12). p -Values show significant differences between NP and P.

Article Snippet: Cultured endometrial tissues were further incubated in the medium with recombinant proteins as follows: bovine CCL2 (RP0027B, Kingfisher Biotech., Inc. St. Paul, MN, USA), human CCL8 (281-CP, R&D Systems, Inc. Minneapolis, MN, USA), bovine CCL11 (RP0071B, Kingfisher Biotech.), human CCL14 (1578-HC, R&D Systems), human CCL16 (TP723266, OriGene Technologies, Inc., Rockville, MD, USA), bovine CXCL10 (RP0079B, Kingfisher Biotech.), bovine tumor necrosis factor-α (TNF; 2279-BT, R&D Systems), human leukemia inhibitory factor (LIF; TP723270, OriGene Technologies), bovine IFNT (1.1 × 10 5 U/mg, generated from HEK293 cells as described previously; Takahashi et al., 2017 [ ]) or supernatant derived from homogenized fetal trophoblast on day 18 of pregnancy (FMP).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Possible Roles of CC- and CXC-Chemokines in Regulating Bovine Endometrial Function during Early Pregnancy

doi: 10.3390/ijms18040742

Figure Lengend Snippet: Localization of CCR1 (binds to CCL8, CCL14, and CCL16), CCR2 (binds to CCL2, CCL8, and CCL16), CCR3 (binds to CCL11), and CXCR3 (binds to CXCL10) in the bovine endometrium and fetal trophoblast obtained from cows in their 18th day of pregnancy. Intensive immunoreactivity was observed in endometrial epithelial cells, glandular epithelial cells, or fetal trophoblast. No positive immunoreactivity was observed in the negative control (Control). Scale bar = 50 µm.

Article Snippet: Cultured endometrial tissues were further incubated in the medium with recombinant proteins as follows: bovine CCL2 (RP0027B, Kingfisher Biotech., Inc. St. Paul, MN, USA), human CCL8 (281-CP, R&D Systems, Inc. Minneapolis, MN, USA), bovine CCL11 (RP0071B, Kingfisher Biotech.), human CCL14 (1578-HC, R&D Systems), human CCL16 (TP723266, OriGene Technologies, Inc., Rockville, MD, USA), bovine CXCL10 (RP0079B, Kingfisher Biotech.), bovine tumor necrosis factor-α (TNF; 2279-BT, R&D Systems), human leukemia inhibitory factor (LIF; TP723270, OriGene Technologies), bovine IFNT (1.1 × 10 5 U/mg, generated from HEK293 cells as described previously; Takahashi et al., 2017 [ ]) or supernatant derived from homogenized fetal trophoblast on day 18 of pregnancy (FMP).

Techniques: Negative Control

Journal: International Journal of Molecular Sciences

Article Title: Possible Roles of CC- and CXC-Chemokines in Regulating Bovine Endometrial Function during Early Pregnancy

doi: 10.3390/ijms18040742

Figure Lengend Snippet: Effects of the supernatant derived from homogenized fetal trophoblast (FMP; 200 ng/mL) and interferon-τ (IFNT; 100 ng/mL) on the mRNA expression of ( a ) CCL2, ( b ) CCL8, ( c ) CCL11, ( d ) CCL14, ( e ) CCL16, and ( f ) CXCL10 in cultured bovine endometrial tissues. Homogenization buffer was added at the control group. Data are means ± SEM of five cows and are expressed as relative ratios of the mRNAs to SUZ12. p -Values show significant differences between treated group and control group.

Article Snippet: Cultured endometrial tissues were further incubated in the medium with recombinant proteins as follows: bovine CCL2 (RP0027B, Kingfisher Biotech., Inc. St. Paul, MN, USA), human CCL8 (281-CP, R&D Systems, Inc. Minneapolis, MN, USA), bovine CCL11 (RP0071B, Kingfisher Biotech.), human CCL14 (1578-HC, R&D Systems), human CCL16 (TP723266, OriGene Technologies, Inc., Rockville, MD, USA), bovine CXCL10 (RP0079B, Kingfisher Biotech.), bovine tumor necrosis factor-α (TNF; 2279-BT, R&D Systems), human leukemia inhibitory factor (LIF; TP723270, OriGene Technologies), bovine IFNT (1.1 × 10 5 U/mg, generated from HEK293 cells as described previously; Takahashi et al., 2017 [ ]) or supernatant derived from homogenized fetal trophoblast on day 18 of pregnancy (FMP).

Techniques: Derivative Assay, Expressing, Cell Culture, Homogenization

Journal: International Journal of Molecular Sciences

Article Title: Possible Roles of CC- and CXC-Chemokines in Regulating Bovine Endometrial Function during Early Pregnancy

doi: 10.3390/ijms18040742

Figure Lengend Snippet: Effects of CCL2, CCL8, CCL11, CCL14, CCL16, and CXCL10 (50 ng/mL each) on the mRNA expression of ( a ) interferon-stimulated gene 15 (ISG15), ( b ) myxovirus-resistance gene 1 (MX1), ( c ) cyclooxygenase 2 (COX2), ( d ) oxytocin receptor (OTR), and ( e ) estrogen receptor α (ESR1) in cultured bovine endometrial tissues. Data are means ± SEM of five cows and are expressed as relative ratios of the mRNAs to SUZ12. p -Values show significant differences between treated group and control group.

Article Snippet: Cultured endometrial tissues were further incubated in the medium with recombinant proteins as follows: bovine CCL2 (RP0027B, Kingfisher Biotech., Inc. St. Paul, MN, USA), human CCL8 (281-CP, R&D Systems, Inc. Minneapolis, MN, USA), bovine CCL11 (RP0071B, Kingfisher Biotech.), human CCL14 (1578-HC, R&D Systems), human CCL16 (TP723266, OriGene Technologies, Inc., Rockville, MD, USA), bovine CXCL10 (RP0079B, Kingfisher Biotech.), bovine tumor necrosis factor-α (TNF; 2279-BT, R&D Systems), human leukemia inhibitory factor (LIF; TP723270, OriGene Technologies), bovine IFNT (1.1 × 10 5 U/mg, generated from HEK293 cells as described previously; Takahashi et al., 2017 [ ]) or supernatant derived from homogenized fetal trophoblast on day 18 of pregnancy (FMP).

Techniques: Expressing, Cell Culture

Journal: International Journal of Molecular Sciences

Article Title: Possible Roles of CC- and CXC-Chemokines in Regulating Bovine Endometrial Function during Early Pregnancy

doi: 10.3390/ijms18040742

Figure Lengend Snippet: Primers used in real-time PCR.

Article Snippet: Cultured endometrial tissues were further incubated in the medium with recombinant proteins as follows: bovine CCL2 (RP0027B, Kingfisher Biotech., Inc. St. Paul, MN, USA), human CCL8 (281-CP, R&D Systems, Inc. Minneapolis, MN, USA), bovine CCL11 (RP0071B, Kingfisher Biotech.), human CCL14 (1578-HC, R&D Systems), human CCL16 (TP723266, OriGene Technologies, Inc., Rockville, MD, USA), bovine CXCL10 (RP0079B, Kingfisher Biotech.), bovine tumor necrosis factor-α (TNF; 2279-BT, R&D Systems), human leukemia inhibitory factor (LIF; TP723270, OriGene Technologies), bovine IFNT (1.1 × 10 5 U/mg, generated from HEK293 cells as described previously; Takahashi et al., 2017 [ ]) or supernatant derived from homogenized fetal trophoblast on day 18 of pregnancy (FMP).

Techniques: Sequencing

Journal: International Journal of Molecular Sciences

Article Title: Possible Roles of CC- and CXC-Chemokines in Regulating Bovine Endometrial Function during Early Pregnancy

doi: 10.3390/ijms18040742

Figure Lengend Snippet: Hypothetical model for inhibition of luteolysis by IFNT and chemokines. Although this model is not concerned with the effects of steroids or growth factors, IFNT, CCL2, CCL8, CCL16, CXCL10, and LIF may block TNF-stimulated-COX2 expression in bovine endometrial cells, leading to the reduction of TNF-induced PGF2α output from the cells. Furthermore, IFNT and CCL16 may stimulate anti-viral activity by up-regulating ISG15 and MX1 expression at the time of maternal recognition in cows. Red and blue arrows show stimulatory and inhibitory actions of each substance, respectively. IFNT may stimulate both CCL8 and CXCL10 production and inhibit CCL14 production from bovine endometrium. Effects of CCL11 on bovine endometrial function are still unclear, although its receptor (CCR3) is expressed in the endometrial epithelial cells.

Article Snippet: Cultured endometrial tissues were further incubated in the medium with recombinant proteins as follows: bovine CCL2 (RP0027B, Kingfisher Biotech., Inc. St. Paul, MN, USA), human CCL8 (281-CP, R&D Systems, Inc. Minneapolis, MN, USA), bovine CCL11 (RP0071B, Kingfisher Biotech.), human CCL14 (1578-HC, R&D Systems), human CCL16 (TP723266, OriGene Technologies, Inc., Rockville, MD, USA), bovine CXCL10 (RP0079B, Kingfisher Biotech.), bovine tumor necrosis factor-α (TNF; 2279-BT, R&D Systems), human leukemia inhibitory factor (LIF; TP723270, OriGene Technologies), bovine IFNT (1.1 × 10 5 U/mg, generated from HEK293 cells as described previously; Takahashi et al., 2017 [ ]) or supernatant derived from homogenized fetal trophoblast on day 18 of pregnancy (FMP).

Techniques: Inhibition, Blocking Assay, Expressing, Activity Assay