met ccl5  (R&D Systems)

Journal: Biomedicines

Article Title: The Chemokine (C-C Motif) Receptor 1 Antagonist BX471 Improves Fluid Resuscitation in Rat Models of Hemorrhagic Shock

doi: 10.3390/biomedicines13051241

Figure Lengend Snippet: Serum CCR1 ligand concentrations (pg/mg) at baseline (t = 0 min) and at the end of the experiment (t = 210 min). Light grey bars: vehicle treatment, n = 7. Dark grey bars: 0.5 μmol/kg BX471 treatment, n = 7. ( A ) CCL3, ( B ) CCL4, ( C ) CCL5, and ( D ) CCL7. *: p < 0.05 vs. baseline (t = 0 min).

Article Snippet: Measurements of Chemokine Concentrations: Serum levels of CCL3, CCL4, CCL5, and CCL7 were measured with commercially available rat enzyme-linked immunosorbent assays (ELISA, Boster Bio, Pleasanton, CA, USA) according to the manufacturer’s instructions.

Techniques:

Journal: Frontiers in Immunology

Article Title: NOX4 modulates breast cancer progression through cancer cell metabolic reprogramming and CD8 + T cell antitumor activity

doi: 10.3389/fimmu.2025.1534936

Figure Lengend Snippet: NOX4 enhanced the CD8 + T cells mediate antitumor effect. (A) TIMER2.0 was used to analysis the correlation between NOX4 expression levels and CD8 + T cell infiltration, Scatter plot showing the correlation between NOX4 expression levels and CD8 + T cell infiltration in breast cancer (BRCA) samples (n=1100). Rho=0.346, p=2.57e-26. (B) TISIDB data: Scatter plot showing the correlation between NOX4 expression and central memory CD8 + T cell (Tcm-CD8) infiltration, effector memory CD8+ T cell (Tem-CD8) in BRCA samples (n=1100). Rho=0.44, p<2.2e-16; Rho=0.171, p<1.24e-6. (C) Representative flow cytometry plots showing the percentage of CD8 + T cells within the CD45 + population in EO771 tumors from NOX4 wild-type (WT) and NOX4 knockout (KO) mice. Bar graph quantifying the percentage of CD8 + cells, showing a significant reduction in CD8 + T cell infiltration in NOX4 KO tumors ***p<0.001. n=6 per group. (D) Representative flow cytometry plots showing IFN-γ and Granzyme B expression in CD8 + T cells from EO771 tumors of NOX4 WT and NOX4 KO mice. Bar graphs quantifying the percentage of CD8 + T cells expressing IFN-γ and Granzyme B, ***p<0.001. n=7 per group. (E) Representative flow cytometry plots showing the percentage of DC cells within the EO771 tumors from NOX4 wild-type (WT) and NOX4 knockout (KO) mice. Bar graph quantifying the percentage of DC cells, **p<0.01. n=7 per group. (F) TISIDB data: Scatter plot showing the correlation between NOX4 expression and CCL11 expression in BRCA samples (n=1100). A significant positive correlation is observed. Rho=0.359, p<2.2e-16. Bar graph showing the concentration of CCL11, CCL5 in the tumor microenvironment, *p<0.05. Data are presented as mean ± SEM from 3 independent experiments. Statistical significance was determined using two-tailed Student’s t-test.

Article Snippet: The quantity of CCL11, CCL5 (Bosterbio) were determined in tumor tissue using ELISA kits according to the manufacturer’s instructions.

Techniques: Expressing, Flow Cytometry, Knock-Out, Concentration Assay, Two Tailed Test

Journal: Scientific Reports

Article Title: New insight into a simple high-yielding method for the production of fully folded and functional recombinant human CCL5

doi: 10.1038/s41598-024-75327-y

Figure Lengend Snippet: Purification of recombinant IH-CCL5 using Shuffle lysY cells. ( a ) Workflow of IH-CCL5 purification, figure generated using BioRender. ( b ) His 6 -SUMO-CCL5 construct representation. ( c ) SDS-PAGE analysis of SUMO-CCL5 post Ni 2+ purification. Lane representation follows lane 1: non-induced total protein (TP), lane 2: non-induced soluble (S), lane 3: induced TP, lane 4: induced S, lane 5: induced insoluble (Ins), lane 6: pre-column, lane 7: His-column flow though, lane 8: His-column wash, lane 9–13: elution fractions F4,6,8,10 and 12. ( d ) SUMO cleavage of His 6 -SUMO-CCL5 construct representation. ( e ) SDS-PAGE analysis post nickel column purification of SUMO cleaved CCL5. Lane 1: His 6 -SUMO-CCL5 pre-cleavage (PrC), lane 2: cleaved CCL5 (C), lane 3: column flow-through (FT) and lane 4: column wash (W).

Article Snippet: Commercial recombinant human RANTES (CCL5) produced in E. Coli (with endotoxin level guaranteed ≤ 1 EU/ug) was purchased from Peprotech.

Techniques: Purification, Recombinant, Generated, Construct, SDS Page, Nickel Column

Journal: Scientific Reports

Article Title: New insight into a simple high-yielding method for the production of fully folded and functional recombinant human CCL5

doi: 10.1038/s41598-024-75327-y

Figure Lengend Snippet: CCL5-mediated activation of CCR5. Ligand binding to CCR5 induces receptor phosphorylation, intracellular signals and calcium flux before removal of ß-arrestin (ß-arr.)-bound surface receptors by internalisation leading to CCR5 downmodulation.

Article Snippet: Commercial recombinant human RANTES (CCL5) produced in E. Coli (with endotoxin level guaranteed ≤ 1 EU/ug) was purchased from Peprotech.

Techniques: Activation Assay, Ligand Binding Assay, Phospho-proteomics

Journal: Scientific Reports

Article Title: New insight into a simple high-yielding method for the production of fully folded and functional recombinant human CCL5

doi: 10.1038/s41598-024-75327-y

Figure Lengend Snippet: IH-CCL5 binding induces CCR5 phosphorylation on CHO-CCR5 cells. IH-CCL5 binding and kinetics of CCR5 phosphorylation using IH-CCL5 over the course of 30 min. ( a ) Flow cytometry analysis of IH-CCL5 binding to CCR5, assessed by loss of anti-CCR5 2D7 signal. Histograms for cells in medium (grey filled) and 100 nM IH-CCL5 (blue solid line) overlayed with no 2D7 (grey dotted line). Bar chart displaying change in specific MFI between medium and IH-CCL5 treated cells. Data shown from a representative experiment. Data analysed with t-test *** P < 0.0002. ( b ) CHO-CCR5 immunoblot after 100 nM IH-CCL5 stimulation for up to 60 min. Anti-CCR5 mAb MC5 (1 µg/mL) was used to detect CCR5 and histone-3 (H3) as a loading control (full-length blot in supplementary Fig. 4). ( c ) Fold change in E11/19-APC signal (phospho-FLOW) over 30 min CCR5 stimulation with 100 nM IH-CCL5 ( n = 3). **** P ≤ 0.0001 two-way ANOVA. Graph symbols medium (open square), IH-CCL5 (filled square) and IH-CCL5/MVC (filled triangle).

Article Snippet: Commercial recombinant human RANTES (CCL5) produced in E. Coli (with endotoxin level guaranteed ≤ 1 EU/ug) was purchased from Peprotech.

Techniques: Binding Assay, Phospho-proteomics, Flow Cytometry, Western Blot, Control

Journal: Scientific Reports

Article Title: New insight into a simple high-yielding method for the production of fully folded and functional recombinant human CCL5

doi: 10.1038/s41598-024-75327-y

Figure Lengend Snippet: In-house CCL5 induced downstream CCR5 signalling activity. Chemokine binding activates downstream signalling that facilitates the release of calcium and cell migration. ( a ) Flow cytometry based experimental workflow for measuring calcium release upon chemokine stimulation. Cells were loaded with Fluo-8 AM (calcium reporter dye) and stimulated with 100 nM chemokine +/- TAK-779 and calcium release was measured using CyAn flow cytometer (FITC). Arrows on the graphs show time at which ( b ) 100 nM commercial CCL5 or ( c ) 100 nM IH-CCL5 were added. Results are from a representative experiment. CCL5 induced activation of CCR5 induces downstream signalling, which is involved in cell migration. ( d ) IH-CCL5 mediated CHO-CCR5 migration was assed using a Transwell migration assay with a 12 μm polycarbonate membrane pore size. ( e ) CHO-CCR5 cells were stimulated with 10 nM IH-CCL5 with or without 800 nM TAK-779. Cell migration was determined as the number of cells that migrated through the membrane filter into lower chamber ( n = 3). One-way ANOVA statistical analysis with Bonferroni test was performed on the data **** P < 0.0001.

Article Snippet: Commercial recombinant human RANTES (CCL5) produced in E. Coli (with endotoxin level guaranteed ≤ 1 EU/ug) was purchased from Peprotech.

Techniques: Activity Assay, Binding Assay, Migration, Flow Cytometry, Activation Assay, Transwell Migration Assay, Membrane, Pore Size

Journal: Scientific Reports

Article Title: New insight into a simple high-yielding method for the production of fully folded and functional recombinant human CCL5

doi: 10.1038/s41598-024-75327-y

Figure Lengend Snippet: IH-CCL5 induced CCR5 downmodulation and internalisation. CCR5 downmodulation was assessed through the loss of anti-CCR5 MC5 binding at 37 o C. ( a , b ) Flow cytometry histogram overlays for MC5 (detected with an anti-mouse A647; APC channel) in different conditions (Medium – Solid grey line with grey fill, 100 nM CCL5 – Blue solid line, 100 nM CCL5 + 800 nM TAK-779 – Red solid line and isotype control – Grey dotted line). ( c ) Change in specific MFI following treatment with the indicated recombinant CCL5 alone (black bars) or with TAK-779 pre-treatment (Grey bars) compared to untreated cells ( n = 3). ** P ≤ 0.0021 and **** P ≤ 0.0001 One-way ANOVA with Bonferroni test. ( d ) Comparison of CCR5 downmodulation induced by IH-CCL5 and commercial CCL5 for CHO-CCR5 cells treated with 100 nM chemokine 1 h at 37 o C ( n = 3). T-test showed no significant difference (ns). ( e ) IH-CCL5 induced CCR5 internalisation using immunofluorescence microscopy. CHO-CCR5 cells were prelabelled with MC5 (5 µg/mL) followed by 100 nM chemokine treatment 0.5 h 37 o C. Secondary anti-mouse A568 4 µg/mL (red) and cells mounted in mowiol containing DAPI (blue). Confocal microscopy images (scale bar at 20 μm) analysed on ImageJ.

Article Snippet: Commercial recombinant human RANTES (CCL5) produced in E. Coli (with endotoxin level guaranteed ≤ 1 EU/ug) was purchased from Peprotech.

Techniques: Binding Assay, Flow Cytometry, Control, Recombinant, Comparison, Immunofluorescence, Microscopy, Confocal Microscopy