ccr4 antibody  (Bio-Techne corporation)

Journal: Science Advances

Article Title: Macrophage-derived chemokine CCL22 establishes local LN-mediated adaptive thermogenesis and energy expenditure

doi: 10.1126/sciadv.adn5229

Figure Lengend Snippet: ( A ) Immunoblots and quantification of CCR4 in SVF cells. SVF cells from 10-week-old male C57BL/6 mice were treated with vehicle or AZD2098 (CCR4 antagonist, 100 nM) for 2 days ( n = 3 per group). ( B ) mRNA expression of Ucp1 , Cd137 , and Tmem26 in beige adipocytes. SVF cells from 10-week-old C57BL/6 male mice [divided into four groups: control, rCCL22 (10 ng/ml), CCR4 inhibitor (AZD2098), and AZD2098 + rCCL22] were first treated for 4 days and then induced to beige adipocytes for 5 days at 31°C. ( C and D ) Immunoblots (C) and quantification (D) of UCP1 ( n = 3 per group). ( E ) Immunofluorescence of UCP1 in beige adipocytes. Scale bars, 25 μm. ( F ) Schematic representation of CCR4 KO mouse model generation by the CRISPR strategy. For 8-week-old mice, male Cas9 mice were iWAT-injected with AAV-sgRNAs-CCR4 [2 × 10 13 genome copies/ml] to generate a CCR4 deletion mouse model (CCR4 −/− ). Enhanced green fluorescent protein (EGFP)–labeled AAV-sgRNAs-CCR4 virus was injected into the left iWAT of mice, and EGFP was not detected in the right iWAT and other tissues, verifying the successful injection of the virus. ( G ) Immunoblots and quantification of CCR4 in the iWAT SVF cells ( n = 3 per group). Eight-week-old male Cas9 mice were injected with vehicle or AAV-sgRNA-CCR4 virus for 2 weeks. ( H to K ) Immunofluorescence of UCP1 and H&E staining in iWAT (H); mRNA expression of Ucp1 , Cd137 , and Tmem26 in iWAT (I); and immunoblots and quantification of UCP1(J and K, respectively) ( n = 3 to 6 per group). Eight-week-old male Cas9 mice were injected with vehicle or AAV-sgRNA-CCR4 virus for 2 weeks. Then, wild-type or CCR4 knockout mice were injected with vehicle or rCCL22 (20 μg/kg per day) for 2 weeks at 6°C. Scale bars, 50 μm. Data information: Results are presented as means ± SEM. [(A), (B), (D), (G), (I), and (K)] * P ≤ 0.05 and ** P < 0.01 by nonpaired Student’s t test. ns, not significant.

Article Snippet: Total protein lysates (20 μg) were immunoblotted with rabbit anti–p-FAK (Y397) (1:1000; ABclonal, #AP0302), rabbit anti-FAK (1:1000; ABclonal, A11195), rabbit anti-p65 antibody (1:1000; Abcam, ab32536), rabbit anti-CCR4 antibody (1:1000; Novus Biological, NB56336SS), rabbit anti-UCP1 antibody (1:500; Abcam, ab23841), rabbit anti-tubulin antibody [1:2000; Cell Signaling Technology (CST), 2146S], mouse anti-CCL22 antibody (1:1000; R&D Systems, MAB439-SP), mouse anti-CD206 antibody (Bio-Rad, MAC2235GA), rat anti-F4/80 (1:500; Abcam, ab6640), rat anti-siglecF (1:500; Novus Biological, NBP1-91149), rabbit anti–IL-13 antibody (1:500; ABclonal, A2089), rabbit anti-p-STAT6 antibody (1:500; ABclonal, AP0456), rabbit anti-STAT6 antibody (1:500; ABclonal, A0755), rabbit anti-Histone3 antibody (1:1000; CST, 4499P), followed by goat anti-rat horseradish peroxidase (HRP)–conjugated secondary antibody (1:5000; ABconal, AS028), anti-rabbit HRP-conjugated secondary antibody (1:5000; CST, 7074S), goat anti-mouse HRP-conjugated secondary antibody (1:5000; CST, 96714S).

Techniques: Western Blot, Expressing, Control, Immunofluorescence, CRISPR, Injection, Labeling, Virus, Staining, Knock-Out

Journal: Science Advances

Article Title: Macrophage-derived chemokine CCL22 establishes local LN-mediated adaptive thermogenesis and energy expenditure

doi: 10.1126/sciadv.adn5229

Figure Lengend Snippet: ( A and B ) Immunoblots and quantification of CCR4 in T reg cells (CD4 + CD25 + CD127 + FoxP3 + ), eosinophils (EO; CD45 + F4/80 + CD11b + SiglecF + ), M2 macrophages (M2; CD45 + CD64 + F4/80 + CD206 + ), dendritic cells (DC; CD11c + I-A/I-E + CD103 + ), SVF cells, and MAs, isolated from five 10-week-old male C57BL/6 mice iWAT by flow cytometry sorting, respectively. ( C and D ) Flow cytometry analysis for eosinophils in iWAT from 10-week-old C57BL/6 male mice receiving sham or LNR for 7 days at 6°C ( n = 4 per group). ( E and F ) Flow cytometry analysis of eosinophils in iWAT from 10-week-old male mice ( n = 4 per group). Littermate control or CCR4 knockout mice were injected with vehicle or rCCL22 (20 μg/kg per day) into bilateral iWAT for 14 days at 6 ° C. ( G ) Immunofluorescence of CCR4 in eosinophils sorted from (E) and (F). Scale bars, 10 μm. ( H to J ) Immunofluorescence (H), immunoblots (I), and quantification (J) of UCP1 in beige adipocytes ( n = 3 per group). Eosinophils from the iWAT SVF cells were sorted, treated with vehicle or rCCL22 (10 ng/ml) for 4 days at 31°C, and subsequently induced to differentiate into beige adipocytes for 5 days. Scale bar, 25 μm. ( K ) Schematic representation picture. Eosinophils (from BMDEs, transferred with vehicle or AAV-sgRNA-CCR4 virus for 3 days) and iWAT SVF cells (sorted out eosinophils) were cocultured for 2 days, treated with vehicle or rCCL22 for 4 days at 31°C and induced to form beige adipocytes for 5 days. SVF cells were obtained from 10-week-old Cas9 male iWAT depots. Eosinophils were obtained from the bone marrow of 10-week-old Cas9 male mice. ( L to N ) Immunofluorescence (L) of UCP1, immunoblots (M), and quantification (N) of CCR4 and UCP1 in eosinophils or beige adipocytes ( n = 3 per group). Scale bars, 25 μm. Data information: Results are presented as means ± SEM. [(D), (F), and (N)] * P ≤ 0.05 by nonpaired Student’s t test.

Article Snippet: Total protein lysates (20 μg) were immunoblotted with rabbit anti–p-FAK (Y397) (1:1000; ABclonal, #AP0302), rabbit anti-FAK (1:1000; ABclonal, A11195), rabbit anti-p65 antibody (1:1000; Abcam, ab32536), rabbit anti-CCR4 antibody (1:1000; Novus Biological, NB56336SS), rabbit anti-UCP1 antibody (1:500; Abcam, ab23841), rabbit anti-tubulin antibody [1:2000; Cell Signaling Technology (CST), 2146S], mouse anti-CCL22 antibody (1:1000; R&D Systems, MAB439-SP), mouse anti-CD206 antibody (Bio-Rad, MAC2235GA), rat anti-F4/80 (1:500; Abcam, ab6640), rat anti-siglecF (1:500; Novus Biological, NBP1-91149), rabbit anti–IL-13 antibody (1:500; ABclonal, A2089), rabbit anti-p-STAT6 antibody (1:500; ABclonal, AP0456), rabbit anti-STAT6 antibody (1:500; ABclonal, A0755), rabbit anti-Histone3 antibody (1:1000; CST, 4499P), followed by goat anti-rat horseradish peroxidase (HRP)–conjugated secondary antibody (1:5000; ABconal, AS028), anti-rabbit HRP-conjugated secondary antibody (1:5000; CST, 7074S), goat anti-mouse HRP-conjugated secondary antibody (1:5000; CST, 96714S).

Techniques: Western Blot, Isolation, Flow Cytometry, Control, Knock-Out, Injection, Immunofluorescence, Virus

Journal: Science Advances

Article Title: Macrophage-derived chemokine CCL22 establishes local LN-mediated adaptive thermogenesis and energy expenditure

doi: 10.1126/sciadv.adn5229

Figure Lengend Snippet: ( A and B ) Immunoblots (A) and quantification (B) of CCR4, p-FAK, and p65 protein in eosinophils cultured with a vehicle, AZD2098 (CCR4 inhibitor, 100 nM), rCCL22 (10 ng/ml), or AZD2098 + rCCL22 (10 ng/ml) for 48 hours ( n = 3 per group). Eosinophils were isolated and induced from BMDEs of 10-week-old male C57BL/6 mice receiving 6°C stimulation for 14 days. ( C ) p65 translocation in eosinophil cells cultured with vehicle, AZD2098 (100 nM), rCCL22 (10 ng/ml), or AZD2098 + rCCL22 (10 ng/ml) for 48 hours ( n = 3 per group). Scale bars, 10 μm. ( D and E ) Immunoblots (D) and quantification (E) of p-FAK and p65 protein in eosinophils cultured with vehicle, PF5733228 (FAK inhibitor, 5 μM), rCCL22 (10 ng/ml), or PF5733228 + rCCL22 (10 ng/ml) for 48 hours. ( F to I ) Immunofluorescence (F) of UCP1, mRNA expression of Ucp1 (G), immunoblots (H), and quantification of UCP1 (I) in beige adipocytes ( n = 3 per group). Eosinophils (treated with vehicle or PF573228 for 2 days) and iWAT SVF cells (without eosinophils) were cocultured for 4 days, and beige adipocytes were then induced for 5 days at 31°C. SVF cells were obtained from 10-week-old C57BL/6 male mouse iWAT. Eosinophils were obtained from the BMDMs of 10-week-old C57BL/6 male mice. Scale bar, 25 μm. Data information: Results are presented as means ± SEM. [(B), (E), (G), and (I)] * P ≤ 0.05 and ** P < 0.01 by nonpaired Student’s t test.

Article Snippet: Total protein lysates (20 μg) were immunoblotted with rabbit anti–p-FAK (Y397) (1:1000; ABclonal, #AP0302), rabbit anti-FAK (1:1000; ABclonal, A11195), rabbit anti-p65 antibody (1:1000; Abcam, ab32536), rabbit anti-CCR4 antibody (1:1000; Novus Biological, NB56336SS), rabbit anti-UCP1 antibody (1:500; Abcam, ab23841), rabbit anti-tubulin antibody [1:2000; Cell Signaling Technology (CST), 2146S], mouse anti-CCL22 antibody (1:1000; R&D Systems, MAB439-SP), mouse anti-CD206 antibody (Bio-Rad, MAC2235GA), rat anti-F4/80 (1:500; Abcam, ab6640), rat anti-siglecF (1:500; Novus Biological, NBP1-91149), rabbit anti–IL-13 antibody (1:500; ABclonal, A2089), rabbit anti-p-STAT6 antibody (1:500; ABclonal, AP0456), rabbit anti-STAT6 antibody (1:500; ABclonal, A0755), rabbit anti-Histone3 antibody (1:1000; CST, 4499P), followed by goat anti-rat horseradish peroxidase (HRP)–conjugated secondary antibody (1:5000; ABconal, AS028), anti-rabbit HRP-conjugated secondary antibody (1:5000; CST, 7074S), goat anti-mouse HRP-conjugated secondary antibody (1:5000; CST, 96714S).

Techniques: Western Blot, Cell Culture, Isolation, Translocation Assay, Immunofluorescence, 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 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