Journal: British Journal of Cancer

Article Title: Chromatin accessibility uncovers KRAS-driven FOSL2 promoting pancreatic ductal adenocarcinoma progression through up-regulation of CCL28

doi: 10.1038/s41416-023-02313-y

Figure Lengend Snippet: a Genomic distribution of FOSL2 CUT&Tag peaks. 5′UTR, 5′ untranslated region. 3′UTR, 3′ untranslated region. b Venn diagram outlining the overlap between FOSL2 target gene associations and 2,143 highly expressed genes in the KPC transcriptome than WT. c GO term analysis of FOSL2-regulated genes. d CUT&Tag signal track showing Ccl28-associated FOSL2 occupancy in KPC mice. e Representative images of IHC staining for CCL28 in WT pancreas, KC, and KPC neoplastic tissues. Scale bar, 100 μm. f OS of PDAC patients grouped by CCL28 mRNA expression. The survival data were obtained from the TCGA database. The high and low expression groups were defined using the median as the group cutoff. g Expression level of CCL28 for main cell types are plotted onto the t-SNE map. The colour key from grey to purple indicates relative expression levels from low to high. The “expression level” was normalised by the logNormalize method in Seurat. h Violin plot showing the global CCL28 expression level from scRNA-seq in the low MUC5AC ductal cell group and high MUC5AC ductal cell group. i RNA expression level of CCL28 in the indicated group in PDAC ductal cells from scRNA-seq data. The statistical test was carried out using the Wilcoxon rank-sum test. j , k CCL28 protein levels in MIA PaCa-2 and PANC-1 pancreatic cancer cells with FOSL2 knockdown ( j ) or in PanO2 cells with FOSL2 overexpression ( k ). For ( j ), the shFOSL2#1 and shFOSL2#2 groups were compared with the shCtrl group, respectively. l , m CCL28 protein in supernatants from MIA PaCa-2 and PANC-1 cells with FOSL2 knockdown ( l ), or from PanO2 cells with FOSL2 overexpression ( m ), as determined by ELISA. For ( l ), the shFOSL2#1 and shFOSL2#2 groups were compared with the shCtrl group, respectively. n , o ChIP analysis of the binding of FOSL2 to the CCL28 upstream in MIA PaCa-2 (N) and PANC-1(O) cells. p FOSL2 motif (top) and scheme of CCL28 upstream luciferase reporter constructs illustrating the wild-type or mutated sequences of potential FOSL2 binding sites (bottom). q , r Luciferase reporter activities of CCL28 upstream or upstream with mutated binding sites in MIA PaCa-2 ( q ) and PANC-1 ( r ) cells with stable FOSL2 knockdown. The shFOSL2#1 and shFOSL2#2 groups were compared with the shCtrl group, respectively. Data are representative of at least three independent experiments and shown as mean ± SEM. * P < 0.05; ** P < 0.01.

Article Snippet: The mice were randomly divided into two groups and were intraperitoneally administered with 3 mg/kg of CCL28 monoclonal antibody (R&D Systems, #MAB533–100) to block CCL28 or isotype IgG once every 3 days.

Techniques: Immunohistochemistry, Expressing, RNA Expression, Knockdown, Over Expression, Enzyme-linked Immunosorbent Assay, Binding Assay, Luciferase, Construct

Journal: British Journal of Cancer

Article Title: Chromatin accessibility uncovers KRAS-driven FOSL2 promoting pancreatic ductal adenocarcinoma progression through up-regulation of CCL28

doi: 10.1038/s41416-023-02313-y

Figure Lengend Snippet: a – d Scheme representing the experimental procedure ( a ), representative tumour image ( b ), tumour weight ( c ), and tumour growth curves ( d ) of C57BL/6 mice injected subcutaneously with Vector and oe-FOSL2 PanO2 cells with treatment of CCL28 neutralising antibody or IgG. e Representative flow staining (left panel) and frequency (right panel) of Treg cells in tumour xenograft of indicated groups. f Representative flow staining (left panel) and frequency (right panel) of CD8 + T cells in tumour xenograft of indicated groups. g The frequency of CD4 + T cells in tumour xenograft of indicated groups. h The level of VEGFA protein in the serum of Vector or oe-FOSL2 mice following administration of anti-CCL28 antibody or IgG, as determined by ELISA. i Representative immunofluorescence staining (left panel) and quantitation (right panel) of mean fluorescence intensity of CD31 in indicated groups. Data are representative shown as mean ± SD or SEM (** P < 0.01; *** P < 0.001; ns not significant).

Article Snippet: The mice were randomly divided into two groups and were intraperitoneally administered with 3 mg/kg of CCL28 monoclonal antibody (R&D Systems, #MAB533–100) to block CCL28 or isotype IgG once every 3 days.

Techniques: Injection, Plasmid Preparation, Staining, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Quantitation Assay, Fluorescence

Journal: British Journal of Cancer

Article Title: Chromatin accessibility uncovers KRAS-driven FOSL2 promoting pancreatic ductal adenocarcinoma progression through up-regulation of CCL28

doi: 10.1038/s41416-023-02313-y

Figure Lengend Snippet: a FOSL2 and CCL28 protein levels in MIA PaCa-2 and PANC-1 cells with KRAS knockdown or in PanO2 cells with mutant KRAS overexpression. b , c FOSL2 mRNA levels in MIA PaCa-2 and PANC-1 cells with KRAS knockdown ( b ) or in PanO2 cells with mutant KRAS overexpression ( c ). For ( b ), the shKRAS#1 and shKRAS#2 groups were compared with the shCtrl group, respectively. d , e CCL28 mRNA levels in MIA PaCa-2 and PANC-1 cells with KRAS knockdown ( d ) or in PanO2 cells with mutant KRAS overexpression ( e ). For ( d ), the shKRAS#1 and shKRAS#2 groups were compared with the shCtrl group, respectively. f , g CCL28 protein in supernatants from MIA PaCa-2 and PANC-1 cells with KRAS knockdown ( f ) or from PanO2 cells with mutant KRAS overexpression ( g ), as determined by ELISA. For ( f ), the shKRAS#1 and shKRAS#2 groups were compared with the shCtrl group, respectively. h , i RT-qPCR showed the FOSL2 ( h ) and CCL28 ( i ) mRNA levels in MIA PaCa-2(G12C), PANC-1(G12D), and PanO2(G12D) cells with or without KRAS/MAPK inhibitors (Selumetinib and SCH772984). j CCL28 protein in supernatants from MIA PaCa-2(G12C), PANC-1(G12D), and PanO2(G12D) cells with or without KRAS/MAPK inhibitors (Selumetinib and SCH772984), as determined by ELISA. k , l Western blotting showed the KRAS, pMEK1/2, tMEK1/2, pERK1/2, tERK1/2, FOSL2, and CCL28 protein levels from MIA PaCa-2(G12C), PANC-1(G12D), and PanO2(G12D) cells with or without KRAS/MAPK inhibitors Selumetinib ( k ) and SCH772984 ( l ). m The mechanistic illustration showed KRAS/MAPK-FOSL2-CCL28-Treg signalling axis in PDAC progression. FOSL2 binds to the proximity of the chemokine gene CCL28 and promotes its transcription and secretion. CCL28 recruits regulatory T cells and leads to tumour immune evasion. Data are representative of at least three independent experiments and shown as mean ± SEM. * P < 0.05; ** P < 0.01.

Article Snippet: The mice were randomly divided into two groups and were intraperitoneally administered with 3 mg/kg of CCL28 monoclonal antibody (R&D Systems, #MAB533–100) to block CCL28 or isotype IgG once every 3 days.

Techniques: Knockdown, Mutagenesis, Over Expression, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Western Blot

Journal: Biology

Article Title: CCL28: A Promising Biomarker for Assessing Salivary Gland Functionality and Maintaining Healthy Oral Environments

doi: 10.3390/biology13030147

Figure Lengend Snippet: CCL28-deficient mice have reduced acidic mucin production in SMG and SLG, with bacterial invasion occurring in their SMG. ( A – F ) SMG and SLG were obtained from WT or CCL28-deficient (CCL28-KO) mice. After preparing the sections, H&E staining ( A , B ) and AB staining ( C , D ) were performed, respectively. Representative images are shown. Scale bars: 100 μm. Relative intensities of AB staining are shown as the means ± SD of results from three independent experiments. *, p < 0.05 ( E , F ). ( G ) Sections of the SMG from WT or CCL28-KO mice were stained with Gram-Hucker’s solution. Representative images are shown. Scale bar: 100 μm (upper panel). The lower panel shows an enlarged image of the dotted area in the upper panel. Scale bar: 10 μm (lower panel). The arrow indicates the gram-positive bacteria.

Article Snippet: The primary antibodies used for immunofluorescence analyses were as follows: Rabbit polyclonal anti-MUC2 (PA5-79702, Invitrogen, Carlsbad, CA, USA) and anti-α-SMA (PA5-87638, Invitrogen); Goat polyclonal anti-CK18 (sc-31700, Santa Cruz Biotechnology, Dallas, TX, USA), anti-mouse CCL28 (AF533, R&D Systems, Minneapolis, MN, USA), and anti-mouse CCR10 (AAM69, Bio-Rad Laboratories, Hercules, CA, USA); Rat monoclonal anti-EpCAM (clone G8.8, BioLegend, San Diego, CA, USA), anti-c-Kit (clone 2B8, BioLegend), and anti-CD31 (clone MEC13.3, BioLegend) antibodies.

Techniques: Staining, Bacteria

Journal: Biology

Article Title: CCL28: A Promising Biomarker for Assessing Salivary Gland Functionality and Maintaining Healthy Oral Environments

doi: 10.3390/biology13030147

Figure Lengend Snippet: CCL28-deficient mice have altered expression of salivary gland markers in the SMG. ( A ) Sections of SMG from WT or CCL28-KO mice were stained for UEA-1 (green) and DNA (blue). Representative images are shown. Scale bar: 40 μm. The right panel shows the same image as the left panel, with the blue signal of the nuclear stain omitted and only the green signal of UEA-1 shown in white. ( B ) Relative intensities of UEA-1 staining are shown as the means ± SD of results from three independent experiments. *, p < 0.05. ( C ) Sections of SMG from WT or CCL28-KO mice were stained for MUC2 (red) and DNA (blue). Representative images are shown. Scale bar: 40 μm. The right panel shows the same image as the left panel, with the blue signal of the nuclear stain omitted and only the red signal of MUC2 shown in white. ( D ) Relative intensities of MUC2 staining are shown as the means ± SD of results from three independent experiments. *, p < 0.05. ( E ) Semi-quantitative RT-PCR for mRNA expression of MUC2, AQP5, α-Amylase, and GAPDH was performed using cDNA prepared from the SMG derived from each of the four WT or CCL28-KO mice. ( F ) The relative expression level of each indicated gene, analyzed using the values obtained by measuring the bands in ( E ), is shown as the means ± SD of results from four independent mice. *, p < 0.05, **, p < 0.01.

Article Snippet: The primary antibodies used for immunofluorescence analyses were as follows: Rabbit polyclonal anti-MUC2 (PA5-79702, Invitrogen, Carlsbad, CA, USA) and anti-α-SMA (PA5-87638, Invitrogen); Goat polyclonal anti-CK18 (sc-31700, Santa Cruz Biotechnology, Dallas, TX, USA), anti-mouse CCL28 (AF533, R&D Systems, Minneapolis, MN, USA), and anti-mouse CCR10 (AAM69, Bio-Rad Laboratories, Hercules, CA, USA); Rat monoclonal anti-EpCAM (clone G8.8, BioLegend, San Diego, CA, USA), anti-c-Kit (clone 2B8, BioLegend), and anti-CD31 (clone MEC13.3, BioLegend) antibodies.

Techniques: Expressing, Staining, Quantitative RT-PCR, Derivative Assay

Journal: Biology

Article Title: CCL28: A Promising Biomarker for Assessing Salivary Gland Functionality and Maintaining Healthy Oral Environments

doi: 10.3390/biology13030147

Figure Lengend Snippet: CCL28-deficient mice lead to structurally defective SMG formation. ( A ) Sections of SMG from WT or CCL28-KO mice were stained with PAS reagent. Representative images are shown. Scale bar: 100 μm. The arrows in the image indicate the duct in which the entire structure was recognized. ( B ) The number of ducts in which the entire structure was recognized, per PAS-stained image of SMG sections from WT or CCL28-KO mice, respectively, was counted as the means ± SD of results from three independent experiments. **, p < 0.01. ( C ) Sections of the SMG from WT or CCL28-KO mice were stained for Phalloidin (F-actin; green) and DNA (blue). Representative images are shown. Scale bar: 40 μm (left panel). The right panel shows an enlarged image of the dotted area in the left panel. Scale bar: 20 μm (right panel). ( D ) Sections of the SMG from WT or CCL28-KO mice were stained for MUC2 (red) and DNA (blue). Representative images are shown. Scale bar: 40 μm (left panel). The right panel shows an enlarged image of the dotted area in the left panel. Scale bar: 10 μm (right panel). ( E ) Sections of the SMG from WT or CCL28-KO mice were stained for CK18 (red) and DNA (blue). Representative images are shown. Scale bar: 40 μm (left panel). The right panel shows an enlarged image of the dotted area in the left panel. Scale bar: 10 μm (right panel). ( F ) Sections of the SMG from WT or CCL28-KO mice were stained for α-SMA (green) and DNA (blue). Representative images are shown. Scale bar: 40 μm. The right panel shows the same image as the left panel, with the blue signal of the nuclear stain omitted and only the green signal of α-SMA shown in white. ( G ) Semi-quantitative RT-PCR for mRNA expression of CK18, α-SMA, and GAPDH was performed using cDNA prepared from the SMG derived from each of the four WT or CCL28-KO mice. ( H ) The relative expression level of each indicated gene, analyzed using the values obtained by measuring the bands in ( G ), is shown as the means ± SD of results from four independent mice. *, p < 0.05.

Article Snippet: The primary antibodies used for immunofluorescence analyses were as follows: Rabbit polyclonal anti-MUC2 (PA5-79702, Invitrogen, Carlsbad, CA, USA) and anti-α-SMA (PA5-87638, Invitrogen); Goat polyclonal anti-CK18 (sc-31700, Santa Cruz Biotechnology, Dallas, TX, USA), anti-mouse CCL28 (AF533, R&D Systems, Minneapolis, MN, USA), and anti-mouse CCR10 (AAM69, Bio-Rad Laboratories, Hercules, CA, USA); Rat monoclonal anti-EpCAM (clone G8.8, BioLegend, San Diego, CA, USA), anti-c-Kit (clone 2B8, BioLegend), and anti-CD31 (clone MEC13.3, BioLegend) antibodies.

Techniques: Staining, Quantitative RT-PCR, Expressing, Derivative Assay

Journal: Biology

Article Title: CCL28: A Promising Biomarker for Assessing Salivary Gland Functionality and Maintaining Healthy Oral Environments

doi: 10.3390/biology13030147

Figure Lengend Snippet: CCL28-deficient mice have reduced expression of stem cell markers in SMG. ( A ) Sections of the SMG from WT or CCL28-KO mice were stained for c-Kit (green) and DNA (blue). Representative images are shown. Scale bar: 40 μm (left panel). The right panel shows an enlarged image of the dotted area in the left panel. Scale bar: 10 μm (right panel). ( B ) Sections of the SMG from WT or CCL28-KO mice were stained for EpCAM (green) and DNA (blue). Representative images are shown. Scale bar: 40 μm (left panel). The right panel shows an enlarged image of the dotted area in the left panel. Scale bar: 10 μm (right panel). ( C ) Semi-quantitative RT-PCR for mRNA expression of c-Kit, EpCAM, and GAPDH was performed using cDNA prepared from the SMG derived from each of the four WT or CCL28-KO mice.

Article Snippet: The primary antibodies used for immunofluorescence analyses were as follows: Rabbit polyclonal anti-MUC2 (PA5-79702, Invitrogen, Carlsbad, CA, USA) and anti-α-SMA (PA5-87638, Invitrogen); Goat polyclonal anti-CK18 (sc-31700, Santa Cruz Biotechnology, Dallas, TX, USA), anti-mouse CCL28 (AF533, R&D Systems, Minneapolis, MN, USA), and anti-mouse CCR10 (AAM69, Bio-Rad Laboratories, Hercules, CA, USA); Rat monoclonal anti-EpCAM (clone G8.8, BioLegend, San Diego, CA, USA), anti-c-Kit (clone 2B8, BioLegend), and anti-CD31 (clone MEC13.3, BioLegend) antibodies.

Techniques: Expressing, Staining, Quantitative RT-PCR, Derivative Assay

Journal: Biology

Article Title: CCL28: A Promising Biomarker for Assessing Salivary Gland Functionality and Maintaining Healthy Oral Environments

doi: 10.3390/biology13030147

Figure Lengend Snippet: CCL28-deficient mice have reduced expression of CD31, a vascular endothelial marker in SMG. ( A ) Sections of the SMG from WT or CCL28-KO mice were stained for CD31 (green) and DNA (blue). Representative images are shown. Scale bar: 40 μm. The right panel shows the same image as the left panel, with the blue signal of the nuclear stain omitted and only the green signal of CD31 shown in white. ( B ) Semi-quantitative RT-PCR for mRNA expression of CD31 and GAPDH was performed using cDNA prepared from the SMG derived from each of the four WT or CCL28-KO mice.

Article Snippet: The primary antibodies used for immunofluorescence analyses were as follows: Rabbit polyclonal anti-MUC2 (PA5-79702, Invitrogen, Carlsbad, CA, USA) and anti-α-SMA (PA5-87638, Invitrogen); Goat polyclonal anti-CK18 (sc-31700, Santa Cruz Biotechnology, Dallas, TX, USA), anti-mouse CCL28 (AF533, R&D Systems, Minneapolis, MN, USA), and anti-mouse CCR10 (AAM69, Bio-Rad Laboratories, Hercules, CA, USA); Rat monoclonal anti-EpCAM (clone G8.8, BioLegend, San Diego, CA, USA), anti-c-Kit (clone 2B8, BioLegend), and anti-CD31 (clone MEC13.3, BioLegend) antibodies.

Techniques: Expressing, Marker, Staining, Quantitative RT-PCR, Derivative Assay

Journal: Renal Failure

Article Title: Assessment of urine CCL2 as a potential diagnostic biomarker for acute kidney injury and septic acute kidney injury in intensive care unit patients

doi: 10.1080/0886022x.2024.2313171

Figure Lengend Snippet: Figure 1. Plasma and urine CCL2 levels between AKI and non-AKI patients at hospital and ICU admission. **p < 0.01; ***p < 0.001; ****p < 0.0001.

Article Snippet: The CCL2 levels in plasma and urine were measured using a commercial human CCL2 ELISA kit (R&D Systems, Bio-Techne, Minneapolis, USA) following the manufacturer’s instructions.

Techniques: Clinical Proteomics

Journal: Renal Failure

Article Title: Assessment of urine CCL2 as a potential diagnostic biomarker for acute kidney injury and septic acute kidney injury in intensive care unit patients

doi: 10.1080/0886022x.2024.2313171

Figure Lengend Snippet: Figure 2. The predictive performance of urine CCL2 for AKI by ROC analysis. (A) ROC curve. (B) AUC and prediction sensitivity and specificity.

Article Snippet: The CCL2 levels in plasma and urine were measured using a commercial human CCL2 ELISA kit (R&D Systems, Bio-Techne, Minneapolis, USA) following the manufacturer’s instructions.

Techniques:

Journal: Renal Failure

Article Title: Assessment of urine CCL2 as a potential diagnostic biomarker for acute kidney injury and septic acute kidney injury in intensive care unit patients

doi: 10.1080/0886022x.2024.2313171

Figure Lengend Snippet: Figure 3. Plasma and urine CCL2 levels between SAKI and non-septic AKI patients at hospital and ICU admission. ***p < 0.001.

Article Snippet: The CCL2 levels in plasma and urine were measured using a commercial human CCL2 ELISA kit (R&D Systems, Bio-Techne, Minneapolis, USA) following the manufacturer’s instructions.

Techniques: Clinical Proteomics

Journal: Renal Failure

Article Title: Assessment of urine CCL2 as a potential diagnostic biomarker for acute kidney injury and septic acute kidney injury in intensive care unit patients

doi: 10.1080/0886022x.2024.2313171

Figure Lengend Snippet: Figure 4. The predictive performance of urine CCL2 for SAKI by ROC analysis. (A) ROC curve. (B) AUC and prediction sensitivity and specificity.

Article Snippet: The CCL2 levels in plasma and urine were measured using a commercial human CCL2 ELISA kit (R&D Systems, Bio-Techne, Minneapolis, USA) following the manufacturer’s instructions.

Techniques:

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: CCL28 has dual roles in mucosal immunity as a chemokine with broad-spectrum antimicrobial activity.

doi: 10.4049/jimmunol.170.3.1452

Figure Lengend Snippet: FIGURE 1. Semiquantitative RT-PCR analysis for expression of CCL28, CCR10, and CCR3 in various mouse tissues. Total RNA samples were prepared from salivary glands, colon, appendix, small intestine with- out Payer’s patches, and Payer’s patches obtained from 12- to 16-wk-old BALB/c mice. RT-PCR was conducted as described in Materials and Methods. Representative results from three separate experiments are shown. Relative signal intensities obtained by normalization with G3PDH are shown in the lower panels as mean SD.

Article Snippet: We used mouse anti-human CCL28 mAb (clone no. 62705) as capturing Ab, biotinylated polyclonal goat anti-human CCL28 (R&D Systems) as detecting Ab, and streptavidin-HRP conjugate (Vector Laboratories) to detect biotinylated second Abs.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: CCL28 has dual roles in mucosal immunity as a chemokine with broad-spectrum antimicrobial activity.

doi: 10.4049/jimmunol.170.3.1452

Figure Lengend Snippet: FIGURE 2. Surface expression of CCR10 and CCR3 and migration to CCL28 of CD3B220low cells isolated from mouse parotid glands. A, Flow cytometric analysis. Single cells were prepared from parotid glands excised from 12- to 16-wk-old BALB/c mice. For detection of cells expressing CCR10, cells were first incubated with CCL27-Fc or control Fc. After washing, cells were incubated with biotin-labeled goat anti- human IgG. After washing, cells were stained with a cocktail of APC-labeled streptavidin, CyChrome-labeled anti-B220, and PE-labeled anti-CD3. For detection of cells expressing CCR3, cells first were incubated with rabbit polyclonal anti-mCCR3 or control rabbit IgG. After washing, cells were incubated with a cocktail of FITC-labeled anti-rabbit IgG, CyChrome-labeled anti-B220, and PE-labeled anti-CD3. Finally, cells were analyzed on FACSCalibur. Representative results from six independent experiments are shown. B, Chemotaxis assay. Single cells prepared from parotid glands were added to the inserts of Transwell plates, with lower wells containing medium without or with indicated concentrations of mCCL2, mCCL28, or mCCL27. After 4 h at 37°C, cells migrated into lower wells were harvested. Original cells and cells migrated into lower wells were stained with PE-labeled anti-CD3 and CyChrome-labeled anti-B220 and were analyzed by flow cytometry in the presence of a known number of counting beads. Filled bars, CD3B220low cells; open bars, CD3B220 cells. The data are mean SD from three separate experiments. C, Plasma cell morphology of CCR10-expressing cells. Single cells prepared from mouse parotid glands were incubated with CCL27-Fc or control Fc. After washing, cells were stained with FITC-labeled goat anti-human IgG. Cells were then placed on glass slides, fixed with methanol, and further stained with May-Gru¨nwald-Giemsa. Cells in the same fields were observed on a fluorescent microscope under UV (Ca) and visible (Cb) lights. No FITC-staining cells were seen by control Fc (data not shown). Magnification, 1000.

Article Snippet: We used mouse anti-human CCL28 mAb (clone no. 62705) as capturing Ab, biotinylated polyclonal goat anti-human CCL28 (R&D Systems) as detecting Ab, and streptavidin-HRP conjugate (Vector Laboratories) to detect biotinylated second Abs.

Techniques: Expressing, Migration, Isolation, Incubation, Control, Labeling, Staining, Chemotaxis Assay, Cytometry, Clinical Proteomics, Microscopy

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: CCL28 has dual roles in mucosal immunity as a chemokine with broad-spectrum antimicrobial activity.

doi: 10.4049/jimmunol.170.3.1452

Figure Lengend Snippet: FIGURE 3. Immunohistochemistry and immunoelectron microscopy of CCL28 in human and mouse salivary glands. Periodate-lysine-4% parafor- maldehyde-prefixed frozen sections of human submandibular gland (A–C, J, and K), mouse parotid gland (D and G), mouse submandibular gland (E and F), and mouse sublingual gland (F and I) were stained with goat anti- human CCL28 (A, B, and J), goat anti-mouse CCL28 (D–F), or normal goat IgG (C, G–I, and K). Immunohistochemistry (A–I): scale bar, 100 m (A–C) and 20 m (D–I); , acinus; , duct. Immunoelectron microscopy (J and K): scale bar, 1 m; arrow, immunoreactive granule; Lu, lumen.

Article Snippet: We used mouse anti-human CCL28 mAb (clone no. 62705) as capturing Ab, biotinylated polyclonal goat anti-human CCL28 (R&D Systems) as detecting Ab, and streptavidin-HRP conjugate (Vector Laboratories) to detect biotinylated second Abs.

Techniques: Immunohistochemistry, Immuno-Electron Microscopy, Staining

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: CCL28 has dual roles in mucosal immunity as a chemokine with broad-spectrum antimicrobial activity.

doi: 10.4049/jimmunol.170.3.1452

Figure Lengend Snippet: FIGURE 4. Exocrine secretion of CCL28. A, Measurement of CCL28 in whole and parotid saliva by ELISA. Whole saliva and parotid secretions were obtained from healthy adult donors. All assays were done in triplicate and mean values were calculated. For details, see Materials and Methods. Whole and parotid saliva samples obtained from the same donors were connected by lines. B, Immunoblot analysis for CCL28. Recombinant CCL28, parotid saliva samples (10 l), and mature milk samples (20 l) were loaded as indicated. For details, see Materials and Methods. Repre- sentative results from two separate experiments are shown.

Article Snippet: We used mouse anti-human CCL28 mAb (clone no. 62705) as capturing Ab, biotinylated polyclonal goat anti-human CCL28 (R&D Systems) as detecting Ab, and streptavidin-HRP conjugate (Vector Laboratories) to detect biotinylated second Abs.

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Recombinant

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: CCL28 has dual roles in mucosal immunity as a chemokine with broad-spectrum antimicrobial activity.

doi: 10.4049/jimmunol.170.3.1452

Figure Lengend Snippet: FIGURE 5. Analysis of the amino acid sequence of human CCL28. A, Amino acid comparison of histatin-5 and CCL28-C. The zinc-binding mo- tifs HExxH and HExxxH are underlined. B, Hydrophobicity plot of hista- tin-5 and CCL28-C. C, A phylogenic tree of chemokines and antimicrobial peptides. MEC/CCL28 and CCL28-C are boxed.

Article Snippet: We used mouse anti-human CCL28 mAb (clone no. 62705) as capturing Ab, biotinylated polyclonal goat anti-human CCL28 (R&D Systems) as detecting Ab, and streptavidin-HRP conjugate (Vector Laboratories) to detect biotinylated second Abs.

Techniques: Sequencing, Comparison, Binding Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: CCL28 has dual roles in mucosal immunity as a chemokine with broad-spectrum antimicrobial activity.

doi: 10.4049/jimmunol.170.3.1452

Figure Lengend Snippet: FIGURE 6. Antimicrobial assay. CCL28, mCCL28, CCL27, CCL28-C, and histatin-5 were examined for antimicrobial activity against C. albicans, P. aeruginosa, and Streptococcus mutans by using the CFU assay. For details, see Materials and Methods. All assays were done in triplicate. Vertical bars indicate SD. Representative results from three separate experiments are shown.

Article Snippet: We used mouse anti-human CCL28 mAb (clone no. 62705) as capturing Ab, biotinylated polyclonal goat anti-human CCL28 (R&D Systems) as detecting Ab, and streptavidin-HRP conjugate (Vector Laboratories) to detect biotinylated second Abs.

Techniques: Activity Assay, Colony-forming Unit Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: CCL28 has dual roles in mucosal immunity as a chemokine with broad-spectrum antimicrobial activity.

doi: 10.4049/jimmunol.170.3.1452

Figure Lengend Snippet: FIGURE 8. Flow cytometric analysis on CCL28 antimicrobial activity. C. albicans were treated with CCL28 as indicated. After washing, cells were stained with FITC-labeled annexin V for 10 min and were washed again. After addition of PI at 2 g/ml, cells were immediately analyzed on FACSCalibur. The representative results of three independent experiments are shown.

Article Snippet: We used mouse anti-human CCL28 mAb (clone no. 62705) as capturing Ab, biotinylated polyclonal goat anti-human CCL28 (R&D Systems) as detecting Ab, and streptavidin-HRP conjugate (Vector Laboratories) to detect biotinylated second Abs.

Techniques: Activity Assay, Staining, Labeling

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: CCL28 has dual roles in mucosal immunity as a chemokine with broad-spectrum antimicrobial activity.

doi: 10.4049/jimmunol.170.3.1452

Figure Lengend Snippet: FIGURE 9. Antimicrobial activity of CCL28 analyzed by scanning electron microscopy. C. albicans (A–F) was mock treated (A and D) or treated with 10 M CCL28 for 30 min (B and E) or 60 min (C and F). P. aeruginosa (G and H) and Streptococcus mutans (I and J) were mock treated (G and I) or treated with 10 M CCL28 for 2 h (H and J). Microbes were immobilized, dried, coated with 3-nm thick platinum-paradium, and observed by a scanning electron microscope. Scale bars: A, 2 m; D, 200 nm; and G, 300 nm.

Article Snippet: We used mouse anti-human CCL28 mAb (clone no. 62705) as capturing Ab, biotinylated polyclonal goat anti-human CCL28 (R&D Systems) as detecting Ab, and streptavidin-HRP conjugate (Vector Laboratories) to detect biotinylated second Abs.

Techniques: Activity Assay, Electron Microscopy, Microscopy

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: CCL28 has dual roles in mucosal immunity as a chemokine with broad-spectrum antimicrobial activity.

doi: 10.4049/jimmunol.170.3.1452

Figure Lengend Snippet: FIGURE 7. Salt-sensitive antimicrobial activity of CCL28. The effects of NaCl concentrations on the antimicrobial activity of CCL28 against C. albicans and P. aeruginosa were examined by using CFU assay. For de- tails, see Materials and Methods. All assays were done in triplicate. Ver- tical bars indicate SD. Representative results from two separate experi- ments are shown.

Article Snippet: We used mouse anti-human CCL28 mAb (clone no. 62705) as capturing Ab, biotinylated polyclonal goat anti-human CCL28 (R&D Systems) as detecting Ab, and streptavidin-HRP conjugate (Vector Laboratories) to detect biotinylated second Abs.

Techniques: Activity Assay, Colony-forming Unit Assay

Journal: Biomolecules

Article Title: Feasibility of Ex Vivo Ligandomics

doi: 10.3390/biom15010145

Figure Lengend Snippet: Flow cytometric analysis of the purity of immunopanned cells. Endothelial cells (ECs) and retinal ganglion cells (RGCs) were immunopanned using anti-CD31 and anti-CD90.2 mAb, respectively, detached by pipetting and analyzed by flow cytometry. ( A ) Immunopanned ECs were labeled with anti-CD31 mAb and quantified with 99.29% purity. ( B ) Human retinal microvascular endothelial cells (HRMVECs) were used as a positive control with 99.89% purity, as labeled with anti-CD31 mAb. ( C ) Immunopanned RGCs were labeled with anti-βIII-tubulin mAb and quantified with 84.80% purity. ( D ) HT22 neuronal cell line was used as a positive control with 97.87% purity, as detected by anti-βIII-tubulin mAb.

Article Snippet: To determine the purity of immunopanned cells, ECs were detached from the plates by pipetting, immunolabeled with DyLight 550-conjugated rat anti-CD31 mAb (1:100 dilution; Novus Biologicals, Centennial, CO, USA; Cat. #NB100-1642R), and incubated for 30 min at 4 °C in the dark.

Techniques: Flow Cytometry, Labeling, Positive Control

Journal: Biomolecules

Article Title: Feasibility of Ex Vivo Ligandomics

doi: 10.3390/biom15010145

Figure Lengend Snippet: Ex vivo Scg3-Phage and VEGF-Phage binding to immunopanned CD31 + ECs. Cells were isolated from the retinas of healthy (white bar) and diabetic (black bar) mice. Control phage ( n = 4 wells) was included as a negative control. ( A ) Binding of Scg3-Phage to ECs in the presence or absence of anti-Scg3 hFab ( n = 5 wells/group). ( B ) Binding of VEGF-Phage to ECs in the presence or absence of aflibercept ( n = 5 wells/group). ± SEM; ns, not significant; t -test.

Article Snippet: To determine the purity of immunopanned cells, ECs were detached from the plates by pipetting, immunolabeled with DyLight 550-conjugated rat anti-CD31 mAb (1:100 dilution; Novus Biologicals, Centennial, CO, USA; Cat. #NB100-1642R), and incubated for 30 min at 4 °C in the dark.

Techniques: Ex Vivo, Binding Assay, Isolation, Control, Negative Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: CCL28 has dual roles in mucosal immunity as a chemokine with broad-spectrum antimicrobial activity.

doi: 10.4049/jimmunol.170.3.1452

Figure Lengend Snippet: FIGURE 1. Semiquantitative RT-PCR analysis for expression of CCL28, CCR10, and CCR3 in various mouse tissues. Total RNA samples were prepared from salivary glands, colon, appendix, small intestine with- out Payer’s patches, and Payer’s patches obtained from 12- to 16-wk-old BALB/c mice. RT-PCR was conducted as described in Materials and Methods. Representative results from three separate experiments are shown. Relative signal intensities obtained by normalization with G3PDH are shown in the lower panels as mean SD.

Article Snippet: Mouse monoclonal anti-human CCL28 (clone no. 62705; IgG1), goat polyclonal anti-human CCL28, biotin-labeled goat polyclonal anti-human CCL28, and goat polyclonal anti-mouse CCL28 were also purchased from R&D Systems.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: CCL28 has dual roles in mucosal immunity as a chemokine with broad-spectrum antimicrobial activity.

doi: 10.4049/jimmunol.170.3.1452

Figure Lengend Snippet: FIGURE 2. Surface expression of CCR10 and CCR3 and migration to CCL28 of CD3B220low cells isolated from mouse parotid glands. A, Flow cytometric analysis. Single cells were prepared from parotid glands excised from 12- to 16-wk-old BALB/c mice. For detection of cells expressing CCR10, cells were first incubated with CCL27-Fc or control Fc. After washing, cells were incubated with biotin-labeled goat anti- human IgG. After washing, cells were stained with a cocktail of APC-labeled streptavidin, CyChrome-labeled anti-B220, and PE-labeled anti-CD3. For detection of cells expressing CCR3, cells first were incubated with rabbit polyclonal anti-mCCR3 or control rabbit IgG. After washing, cells were incubated with a cocktail of FITC-labeled anti-rabbit IgG, CyChrome-labeled anti-B220, and PE-labeled anti-CD3. Finally, cells were analyzed on FACSCalibur. Representative results from six independent experiments are shown. B, Chemotaxis assay. Single cells prepared from parotid glands were added to the inserts of Transwell plates, with lower wells containing medium without or with indicated concentrations of mCCL2, mCCL28, or mCCL27. After 4 h at 37°C, cells migrated into lower wells were harvested. Original cells and cells migrated into lower wells were stained with PE-labeled anti-CD3 and CyChrome-labeled anti-B220 and were analyzed by flow cytometry in the presence of a known number of counting beads. Filled bars, CD3B220low cells; open bars, CD3B220 cells. The data are mean SD from three separate experiments. C, Plasma cell morphology of CCR10-expressing cells. Single cells prepared from mouse parotid glands were incubated with CCL27-Fc or control Fc. After washing, cells were stained with FITC-labeled goat anti-human IgG. Cells were then placed on glass slides, fixed with methanol, and further stained with May-Gru¨nwald-Giemsa. Cells in the same fields were observed on a fluorescent microscope under UV (Ca) and visible (Cb) lights. No FITC-staining cells were seen by control Fc (data not shown). Magnification, 1000.

Article Snippet: Mouse monoclonal anti-human CCL28 (clone no. 62705; IgG1), goat polyclonal anti-human CCL28, biotin-labeled goat polyclonal anti-human CCL28, and goat polyclonal anti-mouse CCL28 were also purchased from R&D Systems.

Techniques: Expressing, Migration, Isolation, Incubation, Control, Labeling, Staining, Chemotaxis Assay, Cytometry, Clinical Proteomics, Microscopy

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: CCL28 has dual roles in mucosal immunity as a chemokine with broad-spectrum antimicrobial activity.

doi: 10.4049/jimmunol.170.3.1452

Figure Lengend Snippet: FIGURE 3. Immunohistochemistry and immunoelectron microscopy of CCL28 in human and mouse salivary glands. Periodate-lysine-4% parafor- maldehyde-prefixed frozen sections of human submandibular gland (A–C, J, and K), mouse parotid gland (D and G), mouse submandibular gland (E and F), and mouse sublingual gland (F and I) were stained with goat anti- human CCL28 (A, B, and J), goat anti-mouse CCL28 (D–F), or normal goat IgG (C, G–I, and K). Immunohistochemistry (A–I): scale bar, 100 m (A–C) and 20 m (D–I); , acinus; , duct. Immunoelectron microscopy (J and K): scale bar, 1 m; arrow, immunoreactive granule; Lu, lumen.

Article Snippet: Mouse monoclonal anti-human CCL28 (clone no. 62705; IgG1), goat polyclonal anti-human CCL28, biotin-labeled goat polyclonal anti-human CCL28, and goat polyclonal anti-mouse CCL28 were also purchased from R&D Systems.

Techniques: Immunohistochemistry, Immuno-Electron Microscopy, Staining

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: CCL28 has dual roles in mucosal immunity as a chemokine with broad-spectrum antimicrobial activity.

doi: 10.4049/jimmunol.170.3.1452

Figure Lengend Snippet: FIGURE 4. Exocrine secretion of CCL28. A, Measurement of CCL28 in whole and parotid saliva by ELISA. Whole saliva and parotid secretions were obtained from healthy adult donors. All assays were done in triplicate and mean values were calculated. For details, see Materials and Methods. Whole and parotid saliva samples obtained from the same donors were connected by lines. B, Immunoblot analysis for CCL28. Recombinant CCL28, parotid saliva samples (10 l), and mature milk samples (20 l) were loaded as indicated. For details, see Materials and Methods. Repre- sentative results from two separate experiments are shown.

Article Snippet: Mouse monoclonal anti-human CCL28 (clone no. 62705; IgG1), goat polyclonal anti-human CCL28, biotin-labeled goat polyclonal anti-human CCL28, and goat polyclonal anti-mouse CCL28 were also purchased from R&D Systems.

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Recombinant

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: CCL28 has dual roles in mucosal immunity as a chemokine with broad-spectrum antimicrobial activity.

doi: 10.4049/jimmunol.170.3.1452

Figure Lengend Snippet: FIGURE 5. Analysis of the amino acid sequence of human CCL28. A, Amino acid comparison of histatin-5 and CCL28-C. The zinc-binding mo- tifs HExxH and HExxxH are underlined. B, Hydrophobicity plot of hista- tin-5 and CCL28-C. C, A phylogenic tree of chemokines and antimicrobial peptides. MEC/CCL28 and CCL28-C are boxed.

Article Snippet: Mouse monoclonal anti-human CCL28 (clone no. 62705; IgG1), goat polyclonal anti-human CCL28, biotin-labeled goat polyclonal anti-human CCL28, and goat polyclonal anti-mouse CCL28 were also purchased from R&D Systems.

Techniques: Sequencing, Comparison, Binding Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: CCL28 has dual roles in mucosal immunity as a chemokine with broad-spectrum antimicrobial activity.

doi: 10.4049/jimmunol.170.3.1452

Figure Lengend Snippet: FIGURE 6. Antimicrobial assay. CCL28, mCCL28, CCL27, CCL28-C, and histatin-5 were examined for antimicrobial activity against C. albicans, P. aeruginosa, and Streptococcus mutans by using the CFU assay. For details, see Materials and Methods. All assays were done in triplicate. Vertical bars indicate SD. Representative results from three separate experiments are shown.

Article Snippet: Mouse monoclonal anti-human CCL28 (clone no. 62705; IgG1), goat polyclonal anti-human CCL28, biotin-labeled goat polyclonal anti-human CCL28, and goat polyclonal anti-mouse CCL28 were also purchased from R&D Systems.

Techniques: Activity Assay, Colony-forming Unit Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: CCL28 has dual roles in mucosal immunity as a chemokine with broad-spectrum antimicrobial activity.

doi: 10.4049/jimmunol.170.3.1452

Figure Lengend Snippet: FIGURE 8. Flow cytometric analysis on CCL28 antimicrobial activity. C. albicans were treated with CCL28 as indicated. After washing, cells were stained with FITC-labeled annexin V for 10 min and were washed again. After addition of PI at 2 g/ml, cells were immediately analyzed on FACSCalibur. The representative results of three independent experiments are shown.

Article Snippet: Mouse monoclonal anti-human CCL28 (clone no. 62705; IgG1), goat polyclonal anti-human CCL28, biotin-labeled goat polyclonal anti-human CCL28, and goat polyclonal anti-mouse CCL28 were also purchased from R&D Systems.

Techniques: Activity Assay, Staining, Labeling

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: CCL28 has dual roles in mucosal immunity as a chemokine with broad-spectrum antimicrobial activity.

doi: 10.4049/jimmunol.170.3.1452

Figure Lengend Snippet: FIGURE 9. Antimicrobial activity of CCL28 analyzed by scanning electron microscopy. C. albicans (A–F) was mock treated (A and D) or treated with 10 M CCL28 for 30 min (B and E) or 60 min (C and F). P. aeruginosa (G and H) and Streptococcus mutans (I and J) were mock treated (G and I) or treated with 10 M CCL28 for 2 h (H and J). Microbes were immobilized, dried, coated with 3-nm thick platinum-paradium, and observed by a scanning electron microscope. Scale bars: A, 2 m; D, 200 nm; and G, 300 nm.

Article Snippet: Mouse monoclonal anti-human CCL28 (clone no. 62705; IgG1), goat polyclonal anti-human CCL28, biotin-labeled goat polyclonal anti-human CCL28, and goat polyclonal anti-mouse CCL28 were also purchased from R&D Systems.

Techniques: Activity Assay, Electron Microscopy, Microscopy

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: CCL28 has dual roles in mucosal immunity as a chemokine with broad-spectrum antimicrobial activity.

doi: 10.4049/jimmunol.170.3.1452

Figure Lengend Snippet: FIGURE 7. Salt-sensitive antimicrobial activity of CCL28. The effects of NaCl concentrations on the antimicrobial activity of CCL28 against C. albicans and P. aeruginosa were examined by using CFU assay. For de- tails, see Materials and Methods. All assays were done in triplicate. Ver- tical bars indicate SD. Representative results from two separate experi- ments are shown.

Article Snippet: Mouse monoclonal anti-human CCL28 (clone no. 62705; IgG1), goat polyclonal anti-human CCL28, biotin-labeled goat polyclonal anti-human CCL28, and goat polyclonal anti-mouse CCL28 were also purchased from R&D Systems.

Techniques: Activity Assay, Colony-forming Unit Assay

Journal: eLife

Article Title: CCL28 modulates neutrophil responses during infection with mucosal pathogens

doi: 10.7554/elife.78206

Figure Lengend Snippet: Figure 2. Absence of CCL28 confers protection in a lethal Acinetobacter pneumonia 959

Article Snippet: 50 nM of recombinant mouse CCL28, CCL11 (R&D Systems), or CXCL1 582 (Peprotech) were placed into the lower compartment of a Transwell chamber.

Techniques:

Journal: eLife

Article Title: CCL28 modulates neutrophil responses during infection with mucosal pathogens

doi: 10.7554/elife.78206

Figure Lengend Snippet: Figure 3. Surface expression of the CCL28 receptors CCR3 and CCR10 on neutrophils 991

Article Snippet: 50 nM of recombinant mouse CCL28, CCL11 (R&D Systems), or CXCL1 582 (Peprotech) were placed into the lower compartment of a Transwell chamber.

Techniques: Expressing

Journal: eLife

Article Title: CCL28 modulates neutrophil responses during infection with mucosal pathogens

doi: 10.7554/elife.78206

Figure Lengend Snippet: Figure 5. CCL28 enhances neutrophil antimicrobial activity. (A) Murine bone marrow 1040

Article Snippet: 50 nM of recombinant mouse CCL28, CCL11 (R&D Systems), or CXCL1 582 (Peprotech) were placed into the lower compartment of a Transwell chamber.

Techniques: Activity Assay

Journal: eLife

Article Title: CCL28 modulates neutrophil responses during infection with mucosal pathogens

doi: 10.7554/elife.78206

Figure Lengend Snippet: Figure 1-figure supplement 2. CCL28 does not confer protection in a Salmonella 1092

Article Snippet: 50 nM of recombinant mouse CCL28, CCL11 (R&D Systems), or CXCL1 582 (Peprotech) were placed into the lower compartment of a Transwell chamber.

Techniques:

Journal: eLife

Article Title: CCL28 modulates neutrophil responses during infection with mucosal pathogens

doi: 10.7554/elife.78206

Figure Lengend Snippet: Figure 1-figure supplement 4. Wild-type and Ccl28-/- mice exhibit similar numbers of B 1122

Article Snippet: 50 nM of recombinant mouse CCL28, CCL11 (R&D Systems), or CXCL1 582 (Peprotech) were placed into the lower compartment of a Transwell chamber.

Techniques: