human ccl28  (R&D Systems)

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Pericytes recruited by CCL28 promote vascular normalization after anti-angiogenesis therapy through RA/RXRA/ANGPT1 pathway in lung adenocarcinoma

doi: 10.1186/s13046-024-03135-3

Figure Lengend Snippet: CCL28 expression is upregulated after anti-angiogenesis therapy by hypoxia-sensitive transcription factor CEBPB in lung adenocarcinoma

Article Snippet: Briefly, pericytes treated with or without recombinant human CCL28 or CCR3 (R&D Systems, MAB155-100) neutralizing antibodies were collected and fixed by adding a cross-linking agent, formaldehyde, to stabilize the interactions between chromatin proteins and DNA.

Techniques: Expressing

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Pericytes recruited by CCL28 promote vascular normalization after anti-angiogenesis therapy through RA/RXRA/ANGPT1 pathway in lung adenocarcinoma

doi: 10.1186/s13046-024-03135-3

Figure Lengend Snippet: Tumor-derived CCL28 recruits pericytes to promote vascular normalization in the tumor microenvironment

Article Snippet: Briefly, pericytes treated with or without recombinant human CCL28 or CCR3 (R&D Systems, MAB155-100) neutralizing antibodies were collected and fixed by adding a cross-linking agent, formaldehyde, to stabilize the interactions between chromatin proteins and DNA.

Techniques: Derivative Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Pericytes recruited by CCL28 promote vascular normalization after anti-angiogenesis therapy through RA/RXRA/ANGPT1 pathway in lung adenocarcinoma

doi: 10.1186/s13046-024-03135-3

Figure Lengend Snippet: Tumor-derived CCL28 promotes the expression of angiopoietin-1 via CCR3 in pericytes

Article Snippet: Briefly, pericytes treated with or without recombinant human CCL28 or CCR3 (R&D Systems, MAB155-100) neutralizing antibodies were collected and fixed by adding a cross-linking agent, formaldehyde, to stabilize the interactions between chromatin proteins and DNA.

Techniques: Derivative Assay, Expressing

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Pericytes recruited by CCL28 promote vascular normalization after anti-angiogenesis therapy through RA/RXRA/ANGPT1 pathway in lung adenocarcinoma

doi: 10.1186/s13046-024-03135-3

Figure Lengend Snippet: Retinoic acid signaling is activated by CCL28 in pericytes through CCR3 A , Volcano plot of changes in metabolic pathways after CCL28 stimulation. B Volcano plot of the enrichment of gene expression after CCL28 stimulation. C Diagram of the metabolic conversion process in the retinoic acid metabolic signaling pathway. D and E Expression of RDH13 and DHRS11 detected by qPCR and western blot with or without exogenous supplement of CCL28. F Correlation of expression of CCL28 with RDH13 in lung adenocarcinoma. G The protein level of DHRS11 and RDH13 stimulated with or without CCL28 and CCR3 neutralizing antibody in pericytes (left) and gray value was calculated(right). H Knockdown efficiency of RDH13 was confirmed by qPCR. I and J Relative expression of RXRα and ANGPT1 after knockdown of RDH13 with or without stimulation of CCL28. K Representative immunofluorescence images of PAN-CK, NG2, CCL28 with DHRS11 or RDH13 or Angiopoietin-1 on biopsy tissues from lung cancer patients (left panel). Scale bar = 100 μm. The correlation between the expression of CCL28 and the levels of DHRS11, RDH13, and angiopoietin-1 (right panel). Data with error bars are shown as mean ± SEM. Each symbol represents data from a replicate. Each panel is a representative experiment of at least three independent biological replicates. *, **, *** represent p < 0.05, p < 0.01 and p < 0.001, respectively. Abbreviation: MFI, Mean fluorescence intensity

Article Snippet: Briefly, pericytes treated with or without recombinant human CCL28 or CCR3 (R&D Systems, MAB155-100) neutralizing antibodies were collected and fixed by adding a cross-linking agent, formaldehyde, to stabilize the interactions between chromatin proteins and DNA.

Techniques: Expressing, Western Blot, Knockdown, Immunofluorescence, Fluorescence

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Pericytes recruited by CCL28 promote vascular normalization after anti-angiogenesis therapy through RA/RXRA/ANGPT1 pathway in lung adenocarcinoma

doi: 10.1186/s13046-024-03135-3

Figure Lengend Snippet: Both CCL28 and retinoic acid could promote vascular normalization in vivo

Article Snippet: Briefly, pericytes treated with or without recombinant human CCL28 or CCR3 (R&D Systems, MAB155-100) neutralizing antibodies were collected and fixed by adding a cross-linking agent, formaldehyde, to stabilize the interactions between chromatin proteins and DNA.

Techniques: In Vivo

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Pericytes recruited by CCL28 promote vascular normalization after anti-angiogenesis therapy through RA/RXRA/ANGPT1 pathway in lung adenocarcinoma

doi: 10.1186/s13046-024-03135-3

Figure Lengend Snippet: CCL28 is involved in bevacizumab-mediated vascular normalization

Article Snippet: Briefly, pericytes treated with or without recombinant human CCL28 or CCR3 (R&D Systems, MAB155-100) neutralizing antibodies were collected and fixed by adding a cross-linking agent, formaldehyde, to stabilize the interactions between chromatin proteins and DNA.

Techniques:

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Pericytes recruited by CCL28 promote vascular normalization after anti-angiogenesis therapy through RA/RXRA/ANGPT1 pathway in lung adenocarcinoma

doi: 10.1186/s13046-024-03135-3

Figure Lengend Snippet: A schematic diagram of tumor microenvironment modulation effects of CCL28

Article Snippet: Briefly, pericytes treated with or without recombinant human CCL28 or CCR3 (R&D Systems, MAB155-100) neutralizing antibodies were collected and fixed by adding a cross-linking agent, formaldehyde, to stabilize the interactions between chromatin proteins and DNA.

Techniques:

Journal: Cell Death & Disease

Article Title: The chemokine receptor CCR10 promotes inflammation-driven hepatocarcinogenesis via PI3K/Akt pathway activation

doi: 10.1038/s41419-018-0267-9

Figure Lengend Snippet: CCR10-transfected HepG2 and LO2 cells were treated with either the CCR10 agonist-ligand CCL28 or the Akt inhibitor A6730. a Activation of the CCL28-CCR10 axis by CCL28 significantly increased Akt phosphorylation, PCNA protein expression, and b relative cell proliferation in both cell lines, while Akt inhibition produced the opposite effects. Relative cell proliferation is defined as the fold-change in proliferation relative to the untreated parent cell line. * P < 0.05 vs. CCR10 group. All values are reported as means ± standard errors of the mean (SEMs)

Article Snippet: For some in vitro experiments, HepG2 and LO2 cell lines were pre-treated with the pro-inflammatory cytokine TNF (concentrations as indicated; R&D Systems, Minneapolis, MN, USA) for 4 h, recombinant human CC chemokine ligand 28 (CCL28) (400 nM; R&D Systems) for 2 h, or the allosteric Akt inhibitor A6730 (10 μM; Sigma, St. Louis, MO, USA) for 2 h . Human CCL28 is a natural ligand-agonist for human CCR10 .

Techniques: Transfection, Activation Assay, Phospho-proteomics, Expressing, Inhibition, Produced

Journal: Cell Death & Disease

Article Title: The chemokine receptor CCR10 promotes inflammation-driven hepatocarcinogenesis via PI3K/Akt pathway activation

doi: 10.1038/s41419-018-0267-9

Figure Lengend Snippet: Following short-term DEN-induced inflammation (10 days after i.p. DEN injection), ( a ) Western blotting analysis showed significantly enhanced TNF protein expression, CCR10 protein expression, PI3K protein expression, Akt phosphorylation, and PCNA protein expression. Knocking-out CCR10 significantly opposed these inflammation-induced effects but did not significantly affect TNF or PI3K protein expression.* P < 0.05 vs. vehicle WT group, † P < 0.05 vs. DEN-treated WT group. b Western blotting analysis of CCR10 protein expression, PI3K protein expression, Akt phosphorylation, and PCNA protein expression in murine liver tissue 6 h after intraperitoneal (i.p.) injection of TNF, which produced significant increases in CCR10 protein expression, PI3K protein expression, Akt phosphorylation, and PCNA protein expression. Pretreatment with the CCR10 agonist-ligand CCL28 significantly increased Akt phosphorylation and PCNA expression levels, while pretreatment with the Akt inhibitor A6730 produced the opposite effects. Neither CCL28 nor A6730 had any significant effect upon CCR10 or PI3K expression. * P < 0.05 vs. vehicle group, † P < 0.05 vs. TNF group. All values are reported as means ± standard errors of the mean (SEMs). n = 12 mice in each group

Article Snippet: For some in vitro experiments, HepG2 and LO2 cell lines were pre-treated with the pro-inflammatory cytokine TNF (concentrations as indicated; R&D Systems, Minneapolis, MN, USA) for 4 h, recombinant human CC chemokine ligand 28 (CCL28) (400 nM; R&D Systems) for 2 h, or the allosteric Akt inhibitor A6730 (10 μM; Sigma, St. Louis, MO, USA) for 2 h . Human CCL28 is a natural ligand-agonist for human CCR10 .

Techniques: Injection, Western Blot, Expressing, Phospho-proteomics, Produced

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Cancer cell chemokines direct chemotaxis of activated stellate cells in pancreatic ductal adenocarcinoma

doi: 10.1038/labinvest.2016.146

Figure Lengend Snippet: A–B ) cDNA was isolated from a panel of human pancreatic cancer cell lines, Panc1, MiaPaCa2, Capan2, Capan1, HPAFII, and Hs766t, and screened by RT-PCR for expression of CCR (A), CXCR, CX 3 CR, or XCR (B) family of receptors. C–D ) The same panel of human cell lines was probed for expression of the known ligands for CCR10 (C) and CXCR6 (D). E ) RT-PCR analysis of patient-derived pancreatic cells (MCW PDAC cell lines) as well as a human pancreatic epithelial nestin-expressing (HPNE) confirmed CXCR6-CXCL16 and CCR10-CCL28 transcript expression. F ) Pancreatic tumor cells derived from the KPC murine model exhibited varying levels of the ligands and receptors. GAPDH and actin were analyzed as loading controls. Regions of the MBL and NRAMP genes were assessed as genomic DNA controls. Positive control was RNA from PBMCs.

Article Snippet: Stimulation or inhibition of migration was mediated via incubation (all treatments were added to the bottom well) with recombinant CCL28 (717-VC, R&D Systems) or polyclonal neutralizing antibodies against CCL28 (AF717, R&D Systems).

Techniques: Isolation, Reverse Transcription Polymerase Chain Reaction, Expressing, Derivative Assay, Positive Control

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Cancer cell chemokines direct chemotaxis of activated stellate cells in pancreatic ductal adenocarcinoma

doi: 10.1038/labinvest.2016.146

Figure Lengend Snippet: A ) Normal pancreatic tissue was sectioned and processed for histopathologic analysis for CCL28, CCR10 and CK19. The arrowhead denotes the epithelial cell lining. B) Tissue from pancreatic tumor was sectioned and processed for immunohistochemical staining with antibodies specific for CCL28, CCR10, CK19, α-SMA as well as H&E, Masson’s trichrome (3C) or Movat’s pentachrome (5C). ‘T’ denotes the tumor cells and ‘S’ denotes the stromal compartments of the tissue section as defined by CK19, α-SMA and trichrome staining. C–D ) Representative stained tissue was scored by an investigator blinded to the tissue or antibody source for staining intensity. * = P ≤ 0.05, ** = P ≤ 0.01, *** = P ≤ 0.001. Tissue staining shown in A and B representative of tissues sections from 14 normal, 12 PanIN, and 12 PDAC patients, based on clinical diagnosis and examination by a board-certified pathologist.

Article Snippet: Stimulation or inhibition of migration was mediated via incubation (all treatments were added to the bottom well) with recombinant CCL28 (717-VC, R&D Systems) or polyclonal neutralizing antibodies against CCL28 (AF717, R&D Systems).

Techniques: Immunohistochemical staining, Staining, Biomarker Discovery

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Cancer cell chemokines direct chemotaxis of activated stellate cells in pancreatic ductal adenocarcinoma

doi: 10.1038/labinvest.2016.146

Figure Lengend Snippet: A) Sections of pancreatitis tissue were processed for immunostaining of CCR10 and CCL28, CXCR6, and CXCL16. B–C) . Scoring for the number of ductal (CK19+) or stromal (SMA+) regions for stain of CXCL16, CXCR6, CCL28 or CCR10. Gray line is the mean staining intensity of normal pancreatic tissue shown in and included as a reference. D ) Parallel tissue sections were processed for immunohistochemistry for CXCL12, CXCR7, CXCR4 and CK19. E ) Quantitative staining intensity of CXCL12, CXCR7 and CXCR4 in CK19+ cells. *** = P ≤ 0.001, **** = P ≤ 0.0001. Tissue staining shown in A and D are representative of tissue specimens from 5–9 individual patients clinically and pathologically diagnosed with pancreatitis.

Article Snippet: Stimulation or inhibition of migration was mediated via incubation (all treatments were added to the bottom well) with recombinant CCL28 (717-VC, R&D Systems) or polyclonal neutralizing antibodies against CCL28 (AF717, R&D Systems).

Techniques: Immunostaining, Staining, Immunohistochemistry

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Cancer cell chemokines direct chemotaxis of activated stellate cells in pancreatic ductal adenocarcinoma

doi: 10.1038/labinvest.2016.146

Figure Lengend Snippet: A ) Sandwich ELISA revealed that the panel of tissue culture pancreatic cancer cells secrete CCL28 when stimulated with 50 ng/mL IFNγ. Levels of CCL28 were undetectable in HPSCs treated with IFNγ. B ) Flow cytometric analysis of HPSCs revealed the cell surface expression of CCR10 (top panel). The mean fluorescence intensity (MFI) was determined for cells stained with the isotype control primary antibody (light gray) and anti-CCR10 antibody (dark gray). C ) CCL28 stimulates chemotaxis. HPSCs were plated in the top well of a transwell insert in serum free media. The bottom well contained media (serum free; SF or full growth; FG). Recombinant CCL28 [30 nM] was added to the bottom or top well as indicated. After 4h, inserts were swabbed, stained with DAPI and enumerated. D ) CCL28-mediated HPSC migration is dose dependent. HPSCs were plated in the top well of a transwell insert. The bottom well contained either full growth media (+; positive control) or serum-free media that either lacked stimulant (-; negative control), or increasing concentrations of recombinant CCL28. E ) HPSC migration is mitigated by an anti-CCL28 neutralizing antibody. The transwell migration setup as above was modified to include the following conditions: no stimulation (NS; negative control), 10 ng/mL TGF-β (positive control) or 30 nM recombinant CCL28. Neutralizing antibody to CCL28 was added to the bottom well for 30 minutes prior to incubation with the cells. F ) CCL28-mediated directional migration of HPSCs is through a G-coupled protein receptor pathway. HPSCs were pretreated with pertussis toxin (PTX) or vehicle, prior to CCL28 stimulation. G) Transcript expression of CCR10 in HPSC cells. RNA was isolated and used as a template for RT-PCR analysis using CCR10 or GAPDH as a loading control. H) CCL28 stimulates chemotactic migration of patient-derived HSPC2 cells. HPSC2 cells were added to a transwell insert and placed in a well containing either serum free medium in the absence (NS) or presence of CCL28 (CCL28) or full growth medium (FG) as a positive control (FG). * = P ≤ 0.05, ** = P ≤ 0.01, *** = P ≤ 0.001.

Article Snippet: Stimulation or inhibition of migration was mediated via incubation (all treatments were added to the bottom well) with recombinant CCL28 (717-VC, R&D Systems) or polyclonal neutralizing antibodies against CCL28 (AF717, R&D Systems).

Techniques: Sandwich ELISA, Expressing, Fluorescence, Staining, Control, Chemotaxis Assay, Recombinant, Migration, Positive Control, Negative Control, Modification, Incubation, Isolation, Reverse Transcription Polymerase Chain Reaction, Derivative Assay

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Cancer cell chemokines direct chemotaxis of activated stellate cells in pancreatic ductal adenocarcinoma

doi: 10.1038/labinvest.2016.146

Figure Lengend Snippet: Three independent HPSC lines were treated with 30 nM CCL28, left untreated (NS), treated with 10 ng/mL TGF-β (C+), treated with SB-431542 [25 µM], a TGF-β1R inhibitor (C-) or treated with vehicle controls (V). A) Representative immunofluorescence images show digital intensity “heat maps” of detected αSMA, corresponding to intensity color bar scale. Scale bars represent 50 µm. B) Semi-quantitative imaging analysis of the mean integrated intensity (Integ Int) of αSMA detection normalized for each HPSC line (HPSC, 2, 3) using the vehicle (V) set as 1 arbitrary unit. HPSC: #, P = 0.0179. ***, P ≤ 0.0002. HPSC2: #, P = 0.0144; ****, P <0.0001; HPSC3, *****, P < 0.0001. C) Representative images of HPSC-derived ECMs produced under experimental conditions as in A and analyzed using Orientation-J plugin of Image-J software. Color tones were normalized using hue values for common mode angle (cyan/green boarded color) visualization as represented on the orientation bar. D) Box and whisker plot of mean percent of fibers distributed within 15° angles from the mode corresponding to the indicated experimental conditions. HPSC: #, P = 0.0304; *, P = 0.028; HPSC2: *, P = 0.0117; HPSC3: C + versus Vs, *, P = 0.0101. No substantial ECM production was obtained from HPSC3 under TGF-β inhibition and was designated as not available (NA).

Article Snippet: Stimulation or inhibition of migration was mediated via incubation (all treatments were added to the bottom well) with recombinant CCL28 (717-VC, R&D Systems) or polyclonal neutralizing antibodies against CCL28 (AF717, R&D Systems).

Techniques: Immunofluorescence, Imaging, Derivative Assay, Produced, Software, Whisker Assay, Inhibition

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Cancer cell chemokines direct chemotaxis of activated stellate cells in pancreatic ductal adenocarcinoma

doi: 10.1038/labinvest.2016.146

Figure Lengend Snippet: Schematic representation of the experimental plan ( A ). Briefly, PDAC epithelial cells were seeded to the bottom well of a transwell dish (1) then stimulated with 50 ng/mL IFNγ to elicit chemokine secretion. HPSCs were plated onto the top chamber of the transwell insert (2) while CCL28 neutralizing antibody was added to the bottom chamber. The insert with HPSCs was added (3) and incubated for 4h prior to (4) fixing, DAPI-staining and cell counting. Stimulation of Panc1 ( B–C ) and MiaPaCa2 ( D–E ) cells by IFNγ resulted in directional migration of HPSCs that was inhibited by the neutralizing CCL28 antibody. HPSC migrate towards PDAC tumor cells and not non-transformed epithelial HPNE cells ( F–G ). Representative images ( C, E ) of 5 independent biological replicates are presented. * = P ≤ 0.05. ** = P ≤ 0.01.

Article Snippet: Stimulation or inhibition of migration was mediated via incubation (all treatments were added to the bottom well) with recombinant CCL28 (717-VC, R&D Systems) or polyclonal neutralizing antibodies against CCL28 (AF717, R&D Systems).

Techniques: Incubation, Staining, Cell Counting, Migration, Transformation Assay

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Cancer cell chemokines direct chemotaxis of activated stellate cells in pancreatic ductal adenocarcinoma

doi: 10.1038/labinvest.2016.146

Figure Lengend Snippet: Normal pancreatic duct epithelial cells and quiescent stromal fibroblasts almost no ligand CCL28 (purple circles) and basal levels of the receptor CCR10 (left panel). In inflammation, such as in pancreatitis, CCL28 expression is increased by cytokines such as IFNγ, which also participate in the activation of stellate cells into cancer-associated fibroblasts. CCL28 directs the migration of stellate cells toward the epithelium (middle panel). CCL28 produced by transformed ductal cells direct the sustained migration of activated stellate cells into the remodeling tumor microenvironment (right panel).

Article Snippet: Stimulation or inhibition of migration was mediated via incubation (all treatments were added to the bottom well) with recombinant CCL28 (717-VC, R&D Systems) or polyclonal neutralizing antibodies against CCL28 (AF717, R&D Systems).

Techniques: Expressing, Activation Assay, Migration, Produced, Transformation Assay

Journal: Biology of reproduction

Article Title: Chemokine (C-C motif) Ligand 28 and Its Receptor CCR10: Expression and Function at the Maternal-Conceptus Interface in Pigs.

doi: 10.1095/biolreprod.116.141903

Figure Lengend Snippet: FIG. 1. Expression of CCL28 in the porcine uterine endometrium during the estrous cycle and pregnancy. A) Real-time RT-PCR analysis of CCL28 mRNA in the uterine endometrium. Endometrial tissue samples from cyclic and pregnant gilts were analyzed by real-time RT-PCR, and the data are reported as expression relative to that detected on Day 12 of the estrous cycle after normalization of the transcript amount to the endogenous RPL7 control. B) In situ hybridization analysis of CCL28 mRNA in the uterine endometrium. Expression of CCL28 mRNA was localized mainly to endometrial GE cells during the estrous cycle and pregnancy. Representative uterine sections from Day 12 of pregnancy, hybridized with a DIG-labeled sense CCL28 cRNA probe (Sense) as a negative control, are shown. D, day; C, estrous cycle; P, pregnancy; LE, luminal epithelium; GE, glandular epithelium; St, stroma; BV, blood vessels. Bars ¼ 200 lm and 100 lm in inset. C) Immunoblot analysis of the CCL28 protein in uterine flushings on Day 12 of the estrous cycle and pregnancy. Uterine flushings were obtained from Day (D) 12 of the estrous cycle (C) and pregnancy (P) and the presence of the CCL28 protein was determined. We loaded rCCL28 as a positive control.

Article Snippet: Fifty-five micrograms of protein from concentrated uterine flushings and 0.5 lg of recombinant human CCL28 protein (rCCL28; R&D Systems) as a control were loaded into and electrophoresed on 12% SDSPAGE gels followed by electrotransfer onto nitrocellulose membranes.

Techniques: Expressing, Quantitative RT-PCR, Control, In Situ Hybridization, Labeling, Negative Control, Western Blot, Positive Control

Journal: Biology of reproduction

Article Title: Chemokine (C-C motif) Ligand 28 and Its Receptor CCR10: Expression and Function at the Maternal-Conceptus Interface in Pigs.

doi: 10.1095/biolreprod.116.141903

Figure Lengend Snippet: FIG. 4. Expression of CCR3 and CCR10 in conceptuses and expression of CCR10 in chorioallantoic membranes. A) RT-PCR analyses of CCL28, CCR3, and CCR10 mRNAs in the uterine endometrium and conceptuses at Days 12 and 15 of pregnancy. Expression of CCL28 and CCR10, but not CCR3, mRNAs was detectable in conceptuses on Days 12 and 15 of pregnancy. RPL7 was used as a positive control. RTase þ/, with (þ) or without () reverse transcriptase; M, molecular marker; D12 Endo, endometrium from Day 12 of pregnancy; D15 Endo, endometrium from Day 15 of pregnancy; D12 Con, Day 12 conceptus; D15 Con, Day 15 conceptus. B) Immunohistochemical analysis of the CCR10 protein in a Day 12 conceptus. CCR10 protein was detected in conceptus trophec- toderm derived from Day 12 of pregnancy. D12P, Day 12 of pregnancy; Tr, trophectoderm; En, endoderm. Bar¼ 50 lm. C) Real-time RT-PCR analysis of the expression of CCR10 mRNA in porcine chorioallantoic tissues during pregnancy. Analysis of the expression of CCR10 mRNA in chorioallantoic tissue samples on Days 30, 60, 90, and 114 of pregnancy revealed that the expression of CCR10 mRNA increased during late pregnancy (linear effect of day, P , 0.01). The data are reported as expression relative to that detected on Day 30 of pregnancy after normalization of the transcript amount to the endogenous RPL7 control, and are presented as least-squares means with SEM.

Article Snippet: Fifty-five micrograms of protein from concentrated uterine flushings and 0.5 lg of recombinant human CCL28 protein (rCCL28; R&D Systems) as a control were loaded into and electrophoresed on 12% SDSPAGE gels followed by electrotransfer onto nitrocellulose membranes.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Positive Control, Reverse Transcription, Marker, Immunohistochemical staining, Derivative Assay, Quantitative RT-PCR, Control

Journal: Biology of reproduction

Article Title: Chemokine (C-C motif) Ligand 28 and Its Receptor CCR10: Expression and Function at the Maternal-Conceptus Interface in Pigs.

doi: 10.1095/biolreprod.116.141903

Figure Lengend Snippet: FIG. 5. Effect of CCL28 on pTr cell proliferation and migration. A) RT-PCR analysis of CCR3 and CCR10 mRNAs in the pTr cell line. The pTr cell line and PBMC as a control expressed CCR10 mRNA, but pTr cells did not express CCR3 mRNA. RPL7 was used as a positive control for RT-PCR. RTase þ/, with (þ) or without () reverse transcriptase; M, molecular marker; D12 Endo, endometrium from Day 12 of pregnancy. B) Effect of rCCL28 on pTr cell proliferation. After serum starvation for 24 h, pTr cells were treated with 0, 0.1, 1, 10, or 100 ng/ml rCCL28 at 378C for 48 h in triplicate. C) Effect of rCCL28 on pTr cell migration. Serum-starved pTr cells were seeded on 8-lm-pore Transwell inserts and treated with different doses (0, 10, or 100 ng/ml) of rCCL28 or 1 lg/ml of rSPP1 as a positive control in triplicate for 12 h. Unmigrated cells on the upper side of the inserts were removed, and cells that had migrated were counted systematically in five nonoverlapping locations. Each independent experiment for cell proliferation and migration was replicated three times. The asterisks denote statistically significant differences (*P , 0.05; **P , 0.01).

Article Snippet: Fifty-five micrograms of protein from concentrated uterine flushings and 0.5 lg of recombinant human CCL28 protein (rCCL28; R&D Systems) as a control were loaded into and electrophoresed on 12% SDSPAGE gels followed by electrotransfer onto nitrocellulose membranes.

Techniques: Migration, Reverse Transcription Polymerase Chain Reaction, Control, Positive Control, Reverse Transcription, Marker