Journal: Frontiers in endocrinology

Article Title: CCL17 and CCL22 chemokines are upregulated in human obesity and play a role in vascular dysfunction.

doi: 10.3389/fendo.2023.1154158

Figure Lengend Snippet: FIGURE 3 CCR4 expression is increased in VCAT from morbid obese patients. (A) Relative quantification of CCR4 mRNA levels. Values are expressed as mean ± SEM (n = 33); (B) Western blot analysis of CCR4 relative protein expression to b-actin in paired and SCAT and VCAT samples. Representative western blots are shown from three different patients (P1–3). Data represent the mean ± SEM of protein densitometry. Comparison between groups were made by Wilcoxon matched-pair signed-rank test. (C) Immunofluorescence representative images showing CCR4 expression in VCAT. Colocalization of CCR4 with CD3 (lymphocytes), CD31 (endothelial cells) and Mac-3 (macrophages) in VCAT. Immunoreactivity was visualized using Alexa Fluor 594 (CCR4, red) and Alexa Fluor 488 (CD31, CD3, Mac-3, green) secondary antibodies. Scale bar, 50 mm. Nuclei were stained with Hoechst (blue).

Article Snippet: Membranes were incubated overnight at 4°C with primary antibodies against human CCR4 (1:200, cat#NB100-56336, Novus Biologicals, Centennial, CO), rabbit anti-human phospho-p44/42 MAPK (ERK1/2) (1:500, cat#4377, Cell Signalling, Danvers, MA), rabbit anti-human p44/42 MAPK (ERK1/2) (1:500, cat#4695, Cell Signalling) and mouse monoclonal antibody against human b-actin (1:1000, cat# SAB1305546, Sigma-Aldrich).

Techniques: Expressing, Western Blot, Comparison, Staining

Journal: Frontiers in endocrinology

Article Title: CCL17 and CCL22 chemokines are upregulated in human obesity and play a role in vascular dysfunction.

doi: 10.3389/fendo.2023.1154158

Figure Lengend Snippet: FIGURE 4 Ex vivo blockade of CCR4 receptor decreases TNFa-induced leukocyte-endothelial cell adhesion in obese patients. (A) Protein CCR4 expression in human aortic endothelial cells (HAEC) (B) Immunofluorescence of representative images showing CCR4 expression in HAEC. Scale bar, 50 mm. (C) Confluent HAECs were stimulated or not with TNFa (20 ng/mL) for 24 h. In parallel, some samples were preincubated with a monoclonal neutralizing antibody against human CCR4 receptor (3 mg/mL). Diluted heparinized whole blood (1:10) from patients (n=16) or healthy controls (n=9) was perfused across endothelial cells monolayers for 5 min at 0.5 dyn/cm2. Values are expressed as mean ± SEM. Representative images show adhered leukocytes to endothelial cell monolayer. Scale bar, 100 mm. Comparisons between groups were made with 1-way ANOVA.

Article Snippet: Membranes were incubated overnight at 4°C with primary antibodies against human CCR4 (1:200, cat#NB100-56336, Novus Biologicals, Centennial, CO), rabbit anti-human phospho-p44/42 MAPK (ERK1/2) (1:500, cat#4377, Cell Signalling, Danvers, MA), rabbit anti-human p44/42 MAPK (ERK1/2) (1:500, cat#4695, Cell Signalling) and mouse monoclonal antibody against human b-actin (1:1000, cat# SAB1305546, Sigma-Aldrich).

Techniques: Ex Vivo, Expressing

Journal: Frontiers in endocrinology

Article Title: CCL17 and CCL22 chemokines are upregulated in human obesity and play a role in vascular dysfunction.

doi: 10.3389/fendo.2023.1154158

Figure Lengend Snippet: FIGURE 5 Neutralizing CCR4 reduces endothelial cell proliferation and morphogenesis. (A) HAEC were incubated with plasma (diluted 1:10 in HBSS) from morbid obese patients or controls subjects for 24h. Some plates were preincubated with a monoclonal blocking antibody against human CCR4 (3 µg/mL). Percentage of proliferating endothelial cells was analysed by BrdU incorporation. Data represent the mean ± SEM of the percentage of BrdU + cells in 3 random fields (40x). (n = 8 control subjects and n = 8 morbidly obese patients). Right panels show representative images of endothelial cell proliferation. Scale bar, 100 mm. (B) Cells cultured in Matrigel were incubated with plasma (diluted 1:10 in HBSS) from obese patients or controls. Phase contrast images were taken after 4 h of incubation and the number of tube-like structures were quantified. Data represent mean ± SEM of the number of tube-like structures or total length of the tubes in 4 random fields (10x). (n = 10 control subjects and n = 10 morbidly obese patients). Right panels show representative images of endothelial cell differentiation on Matrigel. Scale bar, 200 mm.

Article Snippet: Membranes were incubated overnight at 4°C with primary antibodies against human CCR4 (1:200, cat#NB100-56336, Novus Biologicals, Centennial, CO), rabbit anti-human phospho-p44/42 MAPK (ERK1/2) (1:500, cat#4377, Cell Signalling, Danvers, MA), rabbit anti-human p44/42 MAPK (ERK1/2) (1:500, cat#4695, Cell Signalling) and mouse monoclonal antibody against human b-actin (1:1000, cat# SAB1305546, Sigma-Aldrich).

Techniques: Incubation, Clinical Proteomics, Blocking Assay, BrdU Incorporation Assay, Control, Cell Culture, Cell Differentiation

Journal: Frontiers in endocrinology

Article Title: CCL17 and CCL22 chemokines are upregulated in human obesity and play a role in vascular dysfunction.

doi: 10.3389/fendo.2023.1154158

Figure Lengend Snippet: FIGURE 6 Activation of phospho-ERK1/2 MAPK signaling in VCAT from morbid obese patients. (A) Western blot analysis of phospho-ERK1/2 MAPK in paired SCAT and VCAT samples from morbid obese patients (P1-2). Representative Western blot is shown. Data represent the mean ± SEM of protein densitometry (n = 6). Comparisons between groups were made by two-tailed Student´s test. In additional experiments, HAEC were incubated with vehicle (B), hrCCL17, or (C) hrCCL22 (10 ng/ml) for 30 minutes. Some cells were pre-treated with a mouse monoclonal blocking antibody against human CCR4 (3 µg/ml) during 10 minutes before treatment. Representative Western blot is shown. Data represent the mean ± SEM of protein densitometry (n = 7). Comparison between groups were made by one-way ANOVA test.

Article Snippet: Membranes were incubated overnight at 4°C with primary antibodies against human CCR4 (1:200, cat#NB100-56336, Novus Biologicals, Centennial, CO), rabbit anti-human phospho-p44/42 MAPK (ERK1/2) (1:500, cat#4377, Cell Signalling, Danvers, MA), rabbit anti-human p44/42 MAPK (ERK1/2) (1:500, cat#4695, Cell Signalling) and mouse monoclonal antibody against human b-actin (1:1000, cat# SAB1305546, Sigma-Aldrich).

Techniques: Activation Assay, Western Blot, Two Tailed Test, Incubation, Blocking Assay, Comparison

Journal: Indian Journal of Dermatology

Article Title: Evaluation of Lymphocyte Migration to Induced Paederus Dermatitis: An Experimental Study in Rats

doi: 10.4103/ijd.IJD_447_18

Figure Lengend Snippet: The expression of CCR4. The expression of CCR4 was significantly higher in the test group in all internal times

Article Snippet: Following blocking, all sections were subsequently incubated overnight at 4°C with Anti-CD3 Mouse Monoclonal Primary Antibody (1:100 diluted, Santa Cruz, USA), Anti-CCR10 Mouse Monoclonal Primary Antibody (1:200 diluted, Santa Cruz, USA), and Anti-CCR4 Rabbit Polyclonal Primary Antibody (1:100 diluted, Abnova, Taiwan).

Techniques: Expressing

Journal: Indian Journal of Dermatology

Article Title: Evaluation of Lymphocyte Migration to Induced Paederus Dermatitis: An Experimental Study in Rats

doi: 10.4103/ijd.IJD_447_18

Figure Lengend Snippet: The comparison of expression of CD3, CCR4, and CCR10. Based on the results of immunohistochemistry, the expression of CD3, CCR4, and CCR10 in test group at 24, 72, and 120 h compared to the control group showed a significant increase. The peak of expression of all markers was in 72 h after the exposure

Article Snippet: Following blocking, all sections were subsequently incubated overnight at 4°C with Anti-CD3 Mouse Monoclonal Primary Antibody (1:100 diluted, Santa Cruz, USA), Anti-CCR10 Mouse Monoclonal Primary Antibody (1:200 diluted, Santa Cruz, USA), and Anti-CCR4 Rabbit Polyclonal Primary Antibody (1:100 diluted, Abnova, Taiwan).

Techniques: Comparison, Expressing, Immunohistochemistry, Control

Journal: Indian Journal of Dermatology

Article Title: Evaluation of Lymphocyte Migration to Induced Paederus Dermatitis: An Experimental Study in Rats

doi: 10.4103/ijd.IJD_447_18

Figure Lengend Snippet: Immunohistochemistry staining. (a) CD3 expression after 72 h (x200) (b) CCR4 expression after 72 h (x100) (c) CCR10 expression after 72 h (x100) (d) normal skin (x100)

Article Snippet: Following blocking, all sections were subsequently incubated overnight at 4°C with Anti-CD3 Mouse Monoclonal Primary Antibody (1:100 diluted, Santa Cruz, USA), Anti-CCR10 Mouse Monoclonal Primary Antibody (1:200 diluted, Santa Cruz, USA), and Anti-CCR4 Rabbit Polyclonal Primary Antibody (1:100 diluted, Abnova, Taiwan).

Techniques: Immunohistochemistry, Staining, Expressing