Human CCL5/RANTES Antibody

Detection of Human CCL5/RANTES by Simple Western^TM.

Simple Western lane view shows lysates of THP-1 human acute monocytic leukemia cell line untreated (-) or treated (+) with 200nM PMA for 24 hrs and 10ug/mL LPS for 3 hrs, loaded at 0.2 mg/mL. A specific band was detected for CCL5/RANTES at approximately 7 kDa (as indicated) using 10 µg/mL of Goat Anti-Human CCL5/RANTES Antigen Affinity-purified Polyclonal Antibody (Catalog # AF-278-NA) followed by 1:50 dilution of HRP-conjugated Anti-Goat IgG Secondary Antibody (HAF109). This experiment was conducted under reducing conditions and using the 2-40 kDa separation system.

Chemotaxis Induced by CCL5/RANTES and Neutralization by Human CCL5/RANTES Antibody.

Recombinant Human CCL5/RANTES (Catalog # 278-RN) chemoattracts the BaF3 mouse pro-B cell line transfected with human CCR5 in a dose-dependent manner (orange line). The amount of cells that migrated through to the lower chemotaxis chamber was measured by Resazurin (AR002). Chemotaxis elicited by Recombinant Human CCL5/RANTES (0.01 µg/mL) is neutralized (green line) by increasing concentrations of Goat Anti-Human CCL5/RANTES Antigen Affinity-purified Polyclonal Antibody (Catalog # AF-278-NA). The ND₅₀ is typically 0.1-0.4 µg/mL.

Detection of CCL5/RANTES in Human Tonsil.

CCL5/RANTES was detected in immersion fixed paraffin-embedded sections of Human Tonsil using Goat Anti-Human CCL5/RANTES Antigen Affinity-purified Polyclonal Antibody (Catalog # AF-278-NA) at 15 µg/mL for 1 hour at room temperature followed by incubation with the Anti-Goat IgG VisUCyte™ HRP Polymer Antibody (Catalog # VC004). Before incubation with the primary antibody, tissue was subjected to heat-induced epitope retrieval using VisUCyte Antigen Retrieval Reagent-Basic (Catalog # VCTS021). Tissue was stained using DAB (brown) and counterstained with hematoxylin (blue). Specific staining was localized to cytoplasm in lymphocytes. View our protocol for IHC Staining with VisUCyte HRP Polymer Detection Reagents.

Detection of Human CCL5/RANTES by Immunohistochemistry

CCL5-CCR5 axis induced aerobic glycolysis by regulation of AMPK signaling(A) Western blot for AMPK, c-Myc, HIF-1 alpha and Akt in breast cancer cells co-cultured with 15mM lactic acid-activated THP-1 macrophages (ratio 1:1) for 72 h. Results presented were representatives of at least three independent experiments. (B) The expression of AMPK downstream signaling target ACC in breast cancer cells co-cultured as in (A). (C) MDA-MB-231 and MCF-7 cells were transfected with 50 nM AMPK alpha1 siRNA, or pretreated with 10μM compound C for 4 h, and then incubated with 15mM lactic acid-activated THP-1 macrophages (ratio 1:1) for 48 h. The glucose uptake, lactic acid production and ATP levels were detected. (D) The inhibition of AMPK abrogated macrophage-induced EMT in MCF-7 cells. Cells were treated as described in (C). After co-culture, the expression of EMT markers, E-cadherin and vimentin, was measured by western blot. (E) Recombinant human CCL5 induced the phosphorylation of AMPK in MDA-MB-231 and MCF-7/CCR5 cells. 106 cells were treated with 50ng/ml CCL5 for defferent time points as indicated, and phosphorylated AMPK and total AMPK were investigated by western blot. (F) Inhibition of CCR5 in MDA-MB-231 cells significantly attenuated macrophage-induced AMPK phosphorylation. MDA-MB-231 cells were transfected with shRNAs designed against CCR5, or pre-treated with 5μM Maraviroc for 2 h, then co-cultured with 15 mM lactate-activated macrophages as described in (A). After co-culture, the phosphorylation of AMPK was detected by western blot. (G) Expressions of CCL5, CCR5 and p-AMPK in samples obtained from breast cancer patients (n =28). Scale bars represent 50 μm. *, P<0.05; **, P<0.01. Image collected and cropped by CiteAb from the following open publication (https://www.oncotarget.com/lookup/doi/10.18632/oncotarget.22786), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human CCL5/RANTES by Western Blot

Lactate-activated macrophages induced glycolysis through CCL5-CCR5 axis(A) Glucose uptake, lactic acid production and ATP levels in breast cancer cells co-cultured with lactate-activated THP-1 macrophages, with or without 5μg/ml anti-CCL5 neutralizing antibody. The co-culture system was described in Figure 5C. (B) Western blots for glycolytic enzymes in breast cancer cells treated as in (A). (C) MDA-MB-231 cells were transfected with shRNAs designed against CCR5, or pre-treated with 5μM Maraviroc for 2 h, and then subjected to cell co-culture. Glucose uptake, lactic acid production and ATP levels were measured after co-culture. The co-culture system was described in Figure 5C. (D) The protein levels of HK2, PKM2 and LDHA in MDA-MB-231 cells cultured as in (C). (E) Recombinant human CCL5 induced aerobic glycolysis in breast cancer cells. MDA-MB-231 and MCF-7/CCR5 cells were treated with increasing concentrations of CCL5 for 12 h, and glucose uptake, lactic acid production and ATP levels were detected. (F) Western blots for glycolytic enzymes in MDA-MB-231 and MCF-7/CCR5 cells after stimulation with CCL5. *, P<0.05; **, P<0.01. Image collected and cropped by CiteAb from the following open publication (https://www.oncotarget.com/lookup/doi/10.18632/oncotarget.22786), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human Human CCL5/RANTES Antibody by Immunohistochemistry

Lactic acid induced the secretion of CCL5 in human macrophages(A) 3×105 THP-1 macrophages were treated with 15 mM lactate for 24 h, and the mRNA levels of chemokines were measured by quantitative PCR. The growth medium of control macrophages was titrated to pH6.1 using sterile HCl. (B) 3×105 THP-1 macrophages were incubated with different concentrations of lactate for 24 h, and CCL5 gene expression was determined with quantitative PCR. (C) 106 THP-1 macrophages were exposed to increasing concentrations of lactate for 48 h, and the secretion of CCL5 was measured by ELISA. (D) 106 human primary macrophages from breast cancer patients (n=9) were cultured with different concentrations of lactate for 48 h, and CCL5 production was detected. (E) 106 MDA-MB-231 cells were pre-treated with 15μM GSK 2837808A for 2 h, then the media were changed, and cells were cultured for another 24 h. The conditional media (MD-231 CM) were collected and applied to 106 THP-1 macrophages. CCL5 concentrations were detected with ELISA. (F) Immunohistochemical staining of CD68 and CCL5 in tumor adjacent tissues (control) and breast tumors (n=28). Scale bars represent 50 μm. *, P<0.05; **, P<0.01. Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/29299159), licensed under a CC-BY license. Not internally tested by R&D Systems.