recombinant mouse ccl20  (Revvity)

Journal: Journal of Cellular and Molecular Medicine

Article Title: Unraveling CCL20's role by regulating Th17 cell chemotaxis in experimental autoimmune prostatitis

doi: 10.1111/jcmm.18445

Figure Lengend Snippet: The increased levels of CCL20 in the prostate in experimental autoimmune prostatitis mice. (A) Immunohistochemical staining of CCL20 on paraffin‐embedded prostate sections. (B) Semi‐quantitative analysis of CCL20 in Immunohistochemical staining. (C) Relative expression of CCL20 and CCR6 in the prostate measured by RT‐qPCR. (D–F) The expression level of CCL20 and CCR6 in the prostate tissues assessed by Western Blot. (G, H) Quantitative analysis of CCL20 in the serum and prostate of mice determined by ELISA. Representative data from three independent experiments are shown. Data were presented as mean ± SD and were analysed using unpaired, two‐tailed Student's t‐test analysis. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: A portion of the EAP mice received intraperitoneal injections of recombinant mouse CCL20 (rCCL20, 200 μg/mouse; Peprotech, 250–27) or recombinant mouse IL‐17A (100 μg/mouse; Peprotech, 210–17) on a daily basis for 1 week following both the initial and secondary immunizations.

Techniques: Immunohistochemical staining, Staining, Expressing, Quantitative RT-PCR, Western Blot, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: Journal of Cellular and Molecular Medicine

Article Title: Unraveling CCL20's role by regulating Th17 cell chemotaxis in experimental autoimmune prostatitis

doi: 10.1111/jcmm.18445

Figure Lengend Snippet: The recruitment of Th17 cells by CCL20. (A, D) Counting Th17 cells chemoattracted by CCL20 through transwell assay. (B) Immunohistochemical staining of IL‐17A on paraffin‐embedded prostate sections in each group. (C) Assessment of cell viability of Th17 cells after treatment with rCCL20 or anti‐CCL20 neutralizing antibodies. (E) Semi‐quantitative analysis of CCL20 in Immunohistochemical staining. (F) Quantitative analysis of IL‐17A in the serum of mice determined by ELISA. (G, H) Flow cytometry analysis of CD4 + IL‐17A + cells in the prostate. (I–K) The expression level of IL‐17A and CCR6 in the prostate tissues determined by Western Blot. (L) Relative expression of IL‐17A and CCR6 in the prostate measured by RT‐qPCR. Representative data from three independent experiments are shown. (M, N) Quantitative analysis of inflammatory cytokines (IL‐17A, IL‐17F, IL‐22, IFN‐γ, TNF‐α, GM‐CSF) in the serum and prostate of mice determined by ELISA. Data were presented as mean ± SD and were analysed using one‐way anova analysis. ‘N.D.’ Not Determined; ‘ns’ p > 0.05; * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: A portion of the EAP mice received intraperitoneal injections of recombinant mouse CCL20 (rCCL20, 200 μg/mouse; Peprotech, 250–27) or recombinant mouse IL‐17A (100 μg/mouse; Peprotech, 210–17) on a daily basis for 1 week following both the initial and secondary immunizations.

Techniques: Transwell Assay, Immunohistochemical staining, Staining, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Expressing, Western Blot, Quantitative RT-PCR

Journal: Journal of Cellular and Molecular Medicine

Article Title: Unraveling CCL20's role by regulating Th17 cell chemotaxis in experimental autoimmune prostatitis

doi: 10.1111/jcmm.18445

Figure Lengend Snippet: The potential source CCL20 in the prostate of experimental autoimmune prostatitis (EAP) mice. Representative photographs of immunofluorescence staining for CCL20 and markers for (A) CD4 + T cells, (B) macrophages and (C, D) prostatic stromal cells of EAP mice. (E) Quantification of CCL20 immunofluorescence intensity in the experiments of (A–D). Representative data from three independent experiments are shown. Data were presented as mean ± SD and were analysed using one‐way anova analysis. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: A portion of the EAP mice received intraperitoneal injections of recombinant mouse CCL20 (rCCL20, 200 μg/mouse; Peprotech, 250–27) or recombinant mouse IL‐17A (100 μg/mouse; Peprotech, 210–17) on a daily basis for 1 week following both the initial and secondary immunizations.

Techniques: Immunofluorescence, Staining

Journal: Journal of Cellular and Molecular Medicine

Article Title: Unraveling CCL20's role by regulating Th17 cell chemotaxis in experimental autoimmune prostatitis

doi: 10.1111/jcmm.18445

Figure Lengend Snippet: IL‐17A promoted secretion of CCL20 of macrophages via the NF‐κB/MAPK/PI3K pathway. (A, B) Western blot analysis of CCL20 and the phosphorylation of the PI3K (mTOR and AKT) NF‐κB (P65) and MAPK (ERK1/2, P38) pathways in BMDM and RAW264.7 after treatment of LPS and/or rIL‐17. (C, D) Quantitative analysis of CCL20 in the supernatant of BMDM and RAW264.7 determined by ELISA. (E, F) Relative expression of CCL20 in BMDM and RAW264.7 measured by RT‐qPCR after treatment of LPS and/or rIL‐17A. (G, H) Western blot analysis of CCL20 after injection of rIL‐17A or anti‐IL‐17A neutralizing antibodies. (I, J) Quantitative analysis of CCL20 in the serum and prostate of mice determined by ELISA. Representative data from three independent experiments are shown. Data were presented as mean ± SD and were analysed using one‐way anova analysis. ‘ns’ p > 0.05; * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: A portion of the EAP mice received intraperitoneal injections of recombinant mouse CCL20 (rCCL20, 200 μg/mouse; Peprotech, 250–27) or recombinant mouse IL‐17A (100 μg/mouse; Peprotech, 210–17) on a daily basis for 1 week following both the initial and secondary immunizations.

Techniques: Western Blot, Phospho-proteomics, Enzyme-linked Immunosorbent Assay, Expressing, Quantitative RT-PCR, Injection

Journal: Cell Death & Disease

Article Title: Keratinocyte transglutaminase 2 promotes CCR6 + γδT-cell recruitment by upregulating CCL20 in psoriatic inflammation

doi: 10.1038/s41419-020-2495-z

Figure Lengend Snippet: a WT and TG2 −/− mice ( n = 11/group) were treated with IMQ for four consecutive days. Cytokine and chemokine mRNA levels in the back skin epidermis were measured by RT-PCR. b , c Primary keratinocytes prepared from WT and TG2 −/− mice were treated with IMQ (200 μM) or Aldara cream (25 μM) for 3 h. Ccl20 , Il6 , and Cxcl1 mRNA ( b ) and protein levels in the media ( c ) were measured by RT-PCR and a multiplex cytometric bead array, respectively ( n = 3/group). d , e HaCaT cells stably transfected with control and TG2 shRNA were treated with IMQ (200 μM) or Aldara cream (25 μM). IL6 , CXCL8 , and CCL20 mRNA ( d ) and protein levels in the media ( e ) were measured by RT-PCR after 3 h and a multiplex cytometric bead array after 6 h, respectively. Protein levels were normalized to total cellular soluble protein ( n = 3/group). f Human primary keratinocytes stably transfected with control or TG2 shRNA were treated with IMQ (200 μM; n = 3). IL6 , CXCL8 , and CCL20 mRNA levels were measured by RT-PCR after 3 h. Data represent the mean ± SEM ( n = 3/group). * p < 0.05; ** p < 0.01.

Article Snippet: Recombinant mouse CCL20 (BioLegend, 582304), CXCL1 (BioLegend, 573704), or conditioned media collected from WT or TG2 −/− mouse primary keratinocytes treated with IMQ for 24 h with or without 1 μg/ml of anti-CCL20 (R&D Systems, MAB7601) or anti-CXCL1 (R&D Systems, MAB453) neutralizing antibody were placed on the lower chamber.

Techniques: Reverse Transcription Polymerase Chain Reaction, Cream, Multiplex Assay, Stable Transfection, Transfection, Control, shRNA

Journal: Cell Death & Disease

Article Title: Keratinocyte transglutaminase 2 promotes CCR6 + γδT-cell recruitment by upregulating CCL20 in psoriatic inflammation

doi: 10.1038/s41419-020-2495-z

Figure Lengend Snippet: a HaCaT cells stably transfected with control and TG2 shRNA were treated with IMQ, and TG2 mRNA levels were determined by RT-PCR. b HaCaT cells were treated with IMQ in the presence of 5-(biotinamido) pentylamine (BP), and their lysates were subjected to western blot analysis with peroxidase-conjugated streptavidin to detect BP-incorporated proteins. In situ TG activity was measured according to band intensity by ImageJ software ( n = 3/group). c HaCaT cells co-transfected with NF-κB-Luc reporters and phRL-TK control vectors were treated with IMQ for 6 h. NF-κB activity was represented by dual luciferase activity normalized to the control ( n = 3/group). d Schematic representation of WT and mutated CCL20-promoter reporter plasmids. Transcription factor-binding sites are indicated. e HaCaT cells co-transfected with reporter constructs and phRL-TK control vectors for 48 h were treated with 200 μM IMQ for 12 h. CCL20-promoter activity was estimated by normalizing firefly luciferase luminescence to phRL-TK vector Renilla luciferase. Data are relative to the pGL3 control ( n = 4/group) and represent the mean ± SEM. * p < 0.05; ** p < 0.01.

Article Snippet: Recombinant mouse CCL20 (BioLegend, 582304), CXCL1 (BioLegend, 573704), or conditioned media collected from WT or TG2 −/− mouse primary keratinocytes treated with IMQ for 24 h with or without 1 μg/ml of anti-CCL20 (R&D Systems, MAB7601) or anti-CXCL1 (R&D Systems, MAB453) neutralizing antibody were placed on the lower chamber.

Techniques: Stable Transfection, Transfection, Control, shRNA, Reverse Transcription Polymerase Chain Reaction, Western Blot, In Situ, Activity Assay, Software, Luciferase, Binding Assay, Construct, Plasmid Preparation

Journal: Cell Death & Disease

Article Title: Keratinocyte transglutaminase 2 promotes CCR6 + γδT-cell recruitment by upregulating CCL20 in psoriatic inflammation

doi: 10.1038/s41419-020-2495-z

Figure Lengend Snippet: a , b Effect of DMSO- or IMQ-treated WT- or TG2 −/− -CM on CCR6 + γδT-cell ( a ) and CD11b + Ly-6G + cell ( b ) migration according to Transwell migration assays. Flow cytometric analysis of the percentage of cells migrating toward the CM in the presence or absence of anti-CXCL1 ( a ) and anti-CCL20 ( b ) antibodies ( n = 3/group). c , d WT and TG2 −/− mouse-derived CCR6 + γδT-cell ( c ) and CD11b + Ly-6G + cell ( d ) migration in response to recombinant mouse CCL20 and CXCL1, respectively. Data represent the mean ± SEM ( n = 3/group). * p < 0.05; ** p < 0.01. e , f Cells were isolated from the back skin of WT and TG2 −/− mice treated with IMQ for four consecutive days and analyzed by flow cytometry. Representative flow cytometric profiles of CCR6 + γδTCR low cells ( e ) and CD11b high Ly-6G + cells ( f ). The percentage of cells is shown. Data represent the mean ± SEM ( n = 9/group). g Skin sections were immunostained with CCR6 (green)- and IL-17 (red)-specific antibodies. Nuclei were stained with DAPI (blue). Scale bar, 100 μm. h – j Back skin was separated into epidermis and dermis and analyzed by RT-PCR for IL-17-producing γδT-cell markers ( h ) ( Ccr6 and Il23r ), IL17 and related cytokines ( i ) ( Il17a , Il17f , and Il22 ), and myeloid cell-derived cytokines ( j ) ( Il12a and Il23a ). Data represent the mean ± SEM ( n = 11/group). * p < 0.05; ** p < 0.01 vs. WT mice.

Article Snippet: Recombinant mouse CCL20 (BioLegend, 582304), CXCL1 (BioLegend, 573704), or conditioned media collected from WT or TG2 −/− mouse primary keratinocytes treated with IMQ for 24 h with or without 1 μg/ml of anti-CCL20 (R&D Systems, MAB7601) or anti-CXCL1 (R&D Systems, MAB453) neutralizing antibody were placed on the lower chamber.

Techniques: Migration, Derivative Assay, Recombinant, Isolation, Flow Cytometry, Staining, Reverse Transcription Polymerase Chain Reaction

Journal: Cell Death & Disease

Article Title: Keratinocyte transglutaminase 2 promotes CCR6 + γδT-cell recruitment by upregulating CCL20 in psoriatic inflammation

doi: 10.1038/s41419-020-2495-z

Figure Lengend Snippet: a – d Gene-expression profile and TG2 correlation were analyzed using reported microarray data from 64 normal controls (NN) and normal skin lesions (PN) and psoriatic lesions (PP) from 58 psoriatic patients. a TG2 mRNA levels in NN, PN, and PP groups. * p < 0.05; ** p < 0.01; *** p < 0.001. b Correlation between TG2 levels and 23,520 genes was analyzed by Spearman’s rank correlation. The ranking and correlation coefficient of the indicated genes are presented in the plots. Scatter plots of correlation between TG2 expression and IL6, CXCL8 , and CCL20 expression ( c ) and IL17F, IL17A , and IL22 expression ( d ) in NN, PN, and PP groups.

Article Snippet: Recombinant mouse CCL20 (BioLegend, 582304), CXCL1 (BioLegend, 573704), or conditioned media collected from WT or TG2 −/− mouse primary keratinocytes treated with IMQ for 24 h with or without 1 μg/ml of anti-CCL20 (R&D Systems, MAB7601) or anti-CXCL1 (R&D Systems, MAB453) neutralizing antibody were placed on the lower chamber.

Techniques: Gene Expression, Microarray, Expressing

Journal: The international journal of biochemistry & cell biology

Article Title: Leukocyte-endothelial cell interaction is enhanced in podocalyxin-deficient mice.

doi: 10.1016/j.biocel.2018.03.018

Figure Lengend Snippet: Fig. 5. Increased chemokine-mediated adhesion of PerC cells to Podxl-deficient endothelial cells. (A) PerC cells were collected from two control mice treated with intraperitoneal injection of 1 mL 4% thioglycollate for 4 h and labeled with calcein-AM. MLEC cultures from two control or KO mice were incubated in the absence or presence of chemokines CCL20 (20 ng/mL), CCL21 (20 nM), CXCL2 (10 ng/mL), CXCL12 (10 ng/mL), or CXCL13 (50 nM) for 1 h and, then, the medium was removed and 5 × 104 calcein-labeled PerC cells were added to each well and incubated for 20 min, as described in Methods. The results are mean values ± SD of four independent experiments performed in triplicate. (B) Representative experiment and quantification of western blot analysis of CXCL13 bound to MLECs. Cells grown to confluence were incubated with 50 nM CXCL13 and, after exhaustive washing to remove unbound chemokine, Triton lysis buffer was added and the cell lysate containing bound CXCL13 was ana- lyzed by western blot as described in Methods. ns, not significant; *, p < 0.05.

Article Snippet: MLECs from control and Podxl-deficient mice were grown to confluence on 96-well plates and treated with chemokines CCL20 (20 ng/mL, R&D Systems 760-M3), CCL21 (20 nM, Peprotech 250-13), CXCL2 (10 ng/mL, Peprotech 250- 15), CXCL12 (10 ng/mL, Peprotech 250-20A), or CXCL13 (50 nM, Peprotech 250-24) for 1 h. Following chemokine incubation, medium was removed and 5×104 calcein-labeled PerC cells were added to each well and incubated for 20min.

Techniques: Control, Injection, Labeling, Incubation, Western Blot, Lysis