antibodies against human ccr4  (Novus Biologicals)

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: Central-European Journal of Immunology

Article Title: E2F2 stimulates CCR4 expression and activates synovial fibroblast-like cells in rheumatoid arthritis

doi: 10.5114/ceji.2021.105243

Figure Lengend Snippet: PCR primer sequences

Article Snippet: The membranes were incubated overnight at 4 oC with rabbit polyclonal antibody against human E2F2 (ab138515, Abcam, UK), goat polyclonal antibody against human CCR4 (ab1669, Abcam, UK) or rabbit polyclonal antibody against human GAPDH (Good Here, China) at a 1 : 1000 dilution.

Techniques:

Journal: Central-European Journal of Immunology

Article Title: E2F2 stimulates CCR4 expression and activates synovial fibroblast-like cells in rheumatoid arthritis

doi: 10.5114/ceji.2021.105243

Figure Lengend Snippet: mRNA expression of the screened genes by PCR array analysis in RASF. A ) C3AR1, B ) IL1RN, C ) IFNG, D ) TLR7, E ) CCR4 mRNA expression level in RASF with transfection of anti-E2F2 siRNA or Allstars siRNA. F ) CCR4 mRNA expression level in RASF with transfection of E2F2-expressing plasmids or the blank plasmids (mock). * p < 0.05, ns – no statistical significance

Article Snippet: The membranes were incubated overnight at 4 oC with rabbit polyclonal antibody against human E2F2 (ab138515, Abcam, UK), goat polyclonal antibody against human CCR4 (ab1669, Abcam, UK) or rabbit polyclonal antibody against human GAPDH (Good Here, China) at a 1 : 1000 dilution.

Techniques: Expressing, Transfection

Journal: Central-European Journal of Immunology

Article Title: E2F2 stimulates CCR4 expression and activates synovial fibroblast-like cells in rheumatoid arthritis

doi: 10.5114/ceji.2021.105243

Figure Lengend Snippet: CCR4 protein expression in RASF using Western blot analysis. A ) CCR4 protein expression in RASF transfected with anti-E2F2 siRNA. B ) CCR4 expression was normalized to GAPDH expression in the siRNA-transfected RASF. C ) CCR4 expression in RASF transfected with E2F2-expressing plasmids. D ) CCR4 expression was normalized to GAPDH expression in the E2F2-expressing RASF. * p < 0.05

Article Snippet: The membranes were incubated overnight at 4 oC with rabbit polyclonal antibody against human E2F2 (ab138515, Abcam, UK), goat polyclonal antibody against human CCR4 (ab1669, Abcam, UK) or rabbit polyclonal antibody against human GAPDH (Good Here, China) at a 1 : 1000 dilution.

Techniques: Expressing, Western Blot, Transfection

Journal: BMC Immunology

Article Title: Human cerebrospinal fluid contains CD4+ memory T cells expressing gut- or skin-specific trafficking determinants: relevance for immunotherapy

doi: 10.1186/1471-2172-7-14

Figure Lengend Snippet: Multi-color flow cytometry was used to analyze the co-expression of CCR4 with CLA or CXCR3 on memory T cells in paired blood and CSF samples from an individual patient with a non-inflammatory neurological disease (A-D) or MS (E-F). The expression of trafficking determinants was analyzed on CD4+/CD45RO+ memory T cells to account for the different percentages of naïve and memory CD4+ cells in blood (A) and CSF (B). As expected, there was an association between the expression of CCR4 and CLA on CD4+/CD45RO+ T cells in peripheral blood (C). A majority of CLA+ memory T cells co-expressed CCR4 also in the CSF, but some CCR4 staining could be detected on CLA- memory T cells (D). While a large population of CCR4+/CXCR3- cells, which have been demonstrated to be enriched for Th2 cells [27], were present in peripheral blood (E), such cells were rare in the CSF (F).

Article Snippet: We thank Dr J Stine, ICOS corporation, Bothell, WA for generously providing us with the antibody against human CCR4 (clone 328B).

Techniques: Flow Cytometry, Expressing, Staining