antibody for ccl5  (R&D Systems)

Journal: Redox Biology

Article Title: PP4 modulates macrophage-neutrophil crosstalk to restrict CCL5 -driven NETosis in sepsis

doi: 10.1016/j.redox.2026.104093

Figure Lengend Snippet: CCL5 enhances LPS-induced PAD4-dependent NETosis in primary neutrophils. (A) Blood samples were collected from each animal group 24 h after CLP, and plasma concentrations of cfDNA/MPO (NETs) were determined ( n = 10 per group). (B) Confocal images of NETs formation in primary neutrophils were stimulated with LPS (1 mg/ml, 2 h) in the presence or absence of CCL5 (50 ng/ml). Blue, 4′,6-diamidino-2-phenylindole (DAPI); green, myeloperoxidase (MPO). Scale bar = 50 mm. (C) Liver was harvested from each group 24 h post-CLP induction and examined by immunoblotting (IB) with the indicated antibodies. (D) The primary neutrophils isolated from each group were stimulated with LPS with or without CCL5 and examined by IB with the indicated antibodies. The quantification of indicated protein ratio was shown in the right. (E-I) The effect of GSK484 on LPS/CCL5-induced NETs formation. The isolated neutrophils from bone marrow were pre-treated with GSK484 (50 nM, 1 h) and further stimulated with either LPS or CCL5 (n = 3). Sytox Green staining (E), superoxide (2-OH-E+) release (F), mitochondrial ROS (G), total cellular ROS (H) and elastase activity (I) were measured. Results are shown as means ± SE. * p < 0.05; ** p < 0.01 (by a one-way ANOVA).

Article Snippet: A neutralizing antibody for CCL5 (#AF-278-NA) was obtained from R&D Systems (Minneapolis, MN, USA).

Techniques: Clinical Proteomics, Western Blot, Isolation, Staining, Activity Assay

Journal: Redox Biology

Article Title: PP4 modulates macrophage-neutrophil crosstalk to restrict CCL5 -driven NETosis in sepsis

doi: 10.1016/j.redox.2026.104093

Figure Lengend Snippet: Macrophage-derived CCL5 via PP4 drives NETs formation. (A) CCL5 production by isolated BMDMs from WT and PP4 myel−KO mice, stimulated with LPS (1 mg/ml, 24 h), was measured by an ELISA. (B–F) NETs formation (B), mitoROS (C), total cellular ROS (D), elastase activity (E) and immunofluorescence images showing co-localization of myeloperoxidase (MPO, green) and DAPI (blue) (F) in differentiated HL-60 cells treated with conditioned media (CM) from LPS-induced WT and PP4 myel−KO BMDMs, with either IgG control or CCL5-neutralizing antibody (10 ng) for 24 h. All results are presented as the means ± SE. * p < 0.05; ** p < 0.01 (by a one-way ANOVA). Scale bar = 50 mm.

Article Snippet: A neutralizing antibody for CCL5 (#AF-278-NA) was obtained from R&D Systems (Minneapolis, MN, USA).

Techniques: Derivative Assay, Isolation, Enzyme-linked Immunosorbent Assay, Activity Assay, Immunofluorescence, Control

Journal: Redox Biology

Article Title: PP4 modulates macrophage-neutrophil crosstalk to restrict CCL5 -driven NETosis in sepsis

doi: 10.1016/j.redox.2026.104093

Figure Lengend Snippet: PP4 suppresses CCL5 expression through the TBK1/IRF3 signaling pathway. (A) Immunoblotting (IB) of the indicated antibodies in LPS-stimulated BMDM cells isolated from WT and PP4 myel−KO mice. (B) BMDMs were stimulated with LPS (1 mg/ml) and assessed by IB using the indicated antibodies after immunoprecipitation (IP) with anti-PP4. (C) IB of TBK1 and β-actin in BMDMs transfected with empty vector (EV, siNT) or siTBK1. (D) The mRNA levels of IL-6, TNF-α, CCL5 and MIP-1a in BMDMs from PP4 myel−KO mice stimulated with LPS in the presence of siNT or siTBK1. Results were normalized to the expression of ACTB (encoding β-actin) and presented relative to those of untreated cells ( n = 3). (E) BMDMs infected with adenoviruses expressing either an EV (Ad-PGK), HA-PP4 WT (Ad-PP4), or HA-PP4 mutant (Ad-PP4mut) were stimulated with LPS and assessed by IB analysis with the indicated antibodies. (F) CCL5 production by BMDMs transduced as in E and stimulated with LPS for 24 h (n = 3). (G) NETs formation by differentiated HL-60 cells was treated with conditioned media (CM) from LPS-induced either Ad-PGK or Ad-PP4 transduced BMDMs (n = 3). Data are presented as mean ± SE for each group. * p < 0.05; ** p < 0.01 (by a one-way ANOVA).

Article Snippet: A neutralizing antibody for CCL5 (#AF-278-NA) was obtained from R&D Systems (Minneapolis, MN, USA).

Techniques: Expressing, Western Blot, Isolation, Immunoprecipitation, Transfection, Plasmid Preparation, Infection, Mutagenesis

Journal: Redox Biology

Article Title: PP4 modulates macrophage-neutrophil crosstalk to restrict CCL5 -driven NETosis in sepsis

doi: 10.1016/j.redox.2026.104093

Figure Lengend Snippet: LPS induces CCR5 upregulation in neutrophils via ERK1/2 signaling. (A) Neutrophils from WT and PP4 myel−KO mice were isolated 24 h after CLP and analyzed by IB using CCR5 and b-action antibodies. (B) Expression of CCR5 in differentiated HL-60 (dHL60) cells was stimulated with LPS (1 mg/ml, 24 h), with or without the indicated inhibitors. Results were normalized to the expression of ACTB (encoding β-actin) and presented relative to control cells. (C) dHL60 cells treated siNT or siPP4 were stimulated with LPS and subsequently analyzed by IB using the indicated antibodies. (D) IP of anti-PP4 from LPS-stimulated dHL60 cells, followed by IB with antibodies against pERk1/2 or PP4. Total cell lysate (TCL) IB was performed with the indicated antibodies. (E) Neutrophils isolated from PP4 myel−KO mice were pre-treated with PD98059 (20 mM, 1 h) and further stimulated with LPS (1 mg/ml, 2 h). IB was analyzed by using indicated antibodies. (F) siPP4-treated dHL60 cells were stimulated with LPS in the presence or absence of PD98059, followed by IB with indicated antibodies. (G) Neutrophils isolated from PP4 myel−KO mice bone marrow infected with adenoviruses expressing EV (Ad-PGK) or gene encoding HA-PP4 WT (Ad-PP4) or HA-PP4 mutant (Ad-PP4mut) were stimulated with LPS, followed by IB analysis with the indicated antibodies. The quantification of indicated protein ratio was shown in the right. (H and I) NETs formation (H) and elastase activity (I) in isolated neutrophils was measured following LPS in the presence or absence of CCL5. All results are expressed as the means ± SE.* p < 0.05; ** p < 0.01 (by a one-way ANOVA). (n = 3).

Article Snippet: A neutralizing antibody for CCL5 (#AF-278-NA) was obtained from R&D Systems (Minneapolis, MN, USA).

Techniques: Isolation, Expressing, Control, Infection, Mutagenesis, Activity Assay

Journal: Redox Biology

Article Title: PP4 modulates macrophage-neutrophil crosstalk to restrict CCL5 -driven NETosis in sepsis

doi: 10.1016/j.redox.2026.104093

Figure Lengend Snippet: Targeting the CCL5–CCR5 axis attenuates neutrophil hyperactivation. (A) The effect of CCR5 inhibitors on LPS/CCL5-induced NETs formation by using Sytox Green staining. Primary neutrophils isolated from WT and PP4 myel−KO mice were pre-treated with TAK-779 (10 nM, 1hr) or UK-427857 (10 nM, 1hr), and further stimulated with either LPS (1 mg/ml) or CCL5 (50 ng/ml) for 24 h. (B and C) The production of mitoROS (B) and elastase activity (C) by neutrophils as in A, was quantified. (D) Differentiated HL-60 (dHL60) cells treated with siNT or siCCR5 were analyzed by IB using the indicated antibodies. (E-H) NETs formation (E), mitoROS (F), elastase activity (G), and immunofluorescence images of myeloperoxidase (MPO, green) and DAPI (blue) co-localization (H) in dHL60 cells transfected with siNT or siCCR5, followed by LPS stimulation with or without CCL5. All results are presented as the means ± SE.* p < 0.05; ** p < 0.01 (by a one-way ANOVA). Scale bar = 50 mm. (n = 3). (I) Schematic representation of the central role of PP4 in neutrophil recruitment and activation.

Article Snippet: A neutralizing antibody for CCL5 (#AF-278-NA) was obtained from R&D Systems (Minneapolis, MN, USA).

Techniques: Staining, Isolation, Activity Assay, Immunofluorescence, Transfection, Activation Assay

Journal: Redox Biology

Article Title: PP4 modulates macrophage-neutrophil crosstalk to restrict CCL5 -driven NETosis in sepsis

doi: 10.1016/j.redox.2026.104093

Figure Lengend Snippet: CCL5 deficiency reduces NETs formation and improves survival in CLP-induced sepsis. (A) Survival curves of WT and CCL5 KO mice following CLP. (B) Plasma levels of cfDNA/MPO (NETs) in WT or CCL5 KO mice were determined 24 h after CLP. (C-E) NETs formation by Sytox Green staining (C), and the production of mitoROS, total cellular ROS and superoxide release (D), and Elastase activity (E) was measured in differentiated HL-60 cells treated with conditioned media (CM) from LPS-induced WT and CCL5 KO BMDMs in the presence or absence methyl-β-cyclodextrin (MβCD, 5 mM). Data are the mean ± SE for each group. * p < 0.05, ** p < 0.01 (by one-way ANOVA). ( n = 6).

Article Snippet: A neutralizing antibody for CCL5 (#AF-278-NA) was obtained from R&D Systems (Minneapolis, MN, USA).

Techniques: Clinical Proteomics, Staining, Activity Assay

Journal: Cancer Research

Article Title: FADD Activation in Hepatocellular Carcinoma Potentiates CD8 + T-cell Responses and Sensitizes to Immune Checkpoint Inhibitors

doi: 10.1158/0008-5472.CAN-24-3854

Figure Lengend Snippet: FADD in hepatoma cells directs intratumoral CD8 + T-cell infiltration via the CCL5–CCR5 chemotaxis pathway. A, Schematic of the in vivo T-cell migration assay. B, Tumor weights and relative FADD mRNA levels in HepG2-vector and HepG2- FADD tumors 14 days after inoculation ( n = 8). C, Representative IHC images of CD8 in HepG2-vector and HepG2- FADD tumors and quantification thereof ( n = 10). Scale bar, 100 μm. D, Tumor weights and relative FADD mRNA levels in Huh7-sh Ctrl and Huh7-sh FADD tumors 14 days after inoculation ( n = 8). E, Representative IHC images of CD8 in Huh7-sh Ctrl and Huh7-sh FADD tumors and quantification thereof ( n = 10). Scale bar, 100 μm. F, Venn diagram of 521 immune-related genes from and cytokines/chemokines from ImmPort identified 36 overlapping cytokines and chemokines. G, mRNA levels of the 36 cytokines and chemokines in HepG2-vector, HepG2-FADD, Huh7-sh Ctrl , and Huh7-sh FADD cells, as well as tumors generated in A . H, Schematic of NSG-HuPBL humanized mouse model ( n = 8–10). I and J, Representative coimmunofluorescence images of CD8 and CCR5 in HepG2-vector and HepG2- FADD ( I ) and Huh7-sh Ctrl and Huh7-sh FADD tumors ( J ). Hoechst marks cell nuclei. Quantification of CCR5 + CD8 + cell proportions in the indicated groups is also provided. Scale bar, 100 μm. Data are presented as the mean ± SD for at least two (mouse models) or three (Western blotting) independent experiments and analyzed with the paired ( B–E , I and J ) or unpaired ( G ) two-tailed Student t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: Additionally, CCL5 nAbs (R&D Systems, #MAB678-SP) or rCCL5 (R&D Systems, #278-RN-050/CF) were injected into HepG2- FADD or Huh7-sh FADD subcutaneous tumors, followed by T-cell adoptive transfer and IHC staining as described above.

Techniques: Chemotaxis Assay, In Vivo, Cell Migration Assay, Plasmid Preparation, Generated, Western Blot, Two Tailed Test

Journal: Cancer Research

Article Title: FADD Activation in Hepatocellular Carcinoma Potentiates CD8 + T-cell Responses and Sensitizes to Immune Checkpoint Inhibitors

doi: 10.1158/0008-5472.CAN-24-3854

Figure Lengend Snippet: FADD upregulates CCL5 by activating NF-κB transcription in HCC cells. A, Western blotting analysis of p-FADD Ser194 , FADD, p-IκBα Ser32 , IκBα, NF-κB-p50, p-NF-κB-p65 Ser536 , and NF-κB-p65. B, Relative CCL5 mRNA levels in the indicated groups. C, Relative mRNA levels of FADD and CCL5 in HepG2-vector and HepG2-FADD cells treated with siNC, siNF-κB-p50, or siNF-κB-p65. D, ChIP-qPCR analysis of NF-κB-p65 in the CCL5 promoter of HepG2-vector, HepG2- FADD , and HepG2- FADD -S194A stable cells. The CCL5 promoter region for TF binding is also shown. Data are presented as the mean ± SD for at least three (Western blot and qPCR) or two (ChIP-qPCR) independent experiments and analyzed with the unpaired, two-tailed Student t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: Additionally, CCL5 nAbs (R&D Systems, #MAB678-SP) or rCCL5 (R&D Systems, #278-RN-050/CF) were injected into HepG2- FADD or Huh7-sh FADD subcutaneous tumors, followed by T-cell adoptive transfer and IHC staining as described above.

Techniques: Western Blot, Plasmid Preparation, ChIP-qPCR, Binding Assay, Two Tailed Test

Journal: Cancer Research

Article Title: FADD Activation in Hepatocellular Carcinoma Potentiates CD8 + T-cell Responses and Sensitizes to Immune Checkpoint Inhibitors

doi: 10.1158/0008-5472.CAN-24-3854

Figure Lengend Snippet: Phosphorylated FADD promotes NF-κB transcriptional activity via formation of a nuclear p-FADD/SAM68/NF-κB complex in HCC cells. A, Representative co-immunofluorescence images of p-FADD Ser194 (red), FADD (green), and nuclei (DAPI, blue) in HepG2- FADD and HepG2-FADD-S194 stable cell lines. Scale bar, 100 μm. B, Western blotting analysis of p-FADD Ser194 and FADD in the cytoplasmic (Cyto) and nuclear (Nuc) fractions. C, Coomassie brilliant blue staining for co-IP of nuclear proteins pulled down with anti-FADD antibody in HepG2-vector, HepG2-FADD, and HepG2-FADD-S194A stable cell lines. D, Venn diagram of 547 proteins identified through MS analysis in samples from C . E, Correlations of the 110 unique HepG2- FADD nuclear proteins with FADD expression, positive regulation of IκB kinase/NF-κB signaling pathway signatures, and positive regulation of NF-κB TF activity in samples from the TCGA-LIHC dataset. F, Protein–protein interaction network analysis of SAM68 with eight nodes. An average node degree of 4.25 with a median (0.4) level of confidence is required as the minimum required interaction score. G, Co-IP of nuclear FADD and SAM68 in HepG2-vector, HepG2-FADD, and HepG2-FADD-S194A stable cell lines, followed by Western blotting analysis of p-FADD Ser194 , FADD, SAM68, NF-κB-p50, p-NF-κB-p65 Ser536 , and NF-κB-p65. Lamin B serves as the loading control for nuclear lysate input, and IgG is the control for the IP assay. L, ladder. H, Western blotting analysis of SAM68, p-FADD Ser194 , FADD, p-IκBα Ser32 , IκBα, NF-κB-p50, p-NF-κB-p65 Ser536 , and NF-κB-p65. I, Relative mRNA levels of FADD and CCL5 in HepG2-vector and HepG2- FADD cells treated with siNC or siSAM68 (#1 and #2). J, Co-IP of nuclear FADD and SAM68 from HepG2-vector, HepG2- FADD , and HepG2-FADD treated with siSAM68 (#2), followed by Western blotting analysis of p-FADD Ser194 , FADD, SAM68, NF-κB-p50, p-NF-κB-p65 Ser536 , and NF-κB-p65. Histone H3 served as the nuclear lysate input loading control, and IgG is the IP assay control. K, Relative induction of NF-κB luciferase in HepG2-vector and HepG2- FADD cells treated with siNC or siSAM68 (#2). Data are presented as the mean ± SD for at least three independent experiments (coimmunofluorescence, Western blotting, and co-IP) and analyzed with the unpaired, two-tailed Student t test. Two-tailed Pearson correlation was used to compute the correlation between variables. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: Additionally, CCL5 nAbs (R&D Systems, #MAB678-SP) or rCCL5 (R&D Systems, #278-RN-050/CF) were injected into HepG2- FADD or Huh7-sh FADD subcutaneous tumors, followed by T-cell adoptive transfer and IHC staining as described above.

Techniques: Activity Assay, Immunofluorescence, Stable Transfection, Western Blot, Staining, Co-Immunoprecipitation Assay, Plasmid Preparation, Expressing, Control, Luciferase, Two Tailed Test

Journal: Cancer Research

Article Title: FADD Activation in Hepatocellular Carcinoma Potentiates CD8 + T-cell Responses and Sensitizes to Immune Checkpoint Inhibitors

doi: 10.1158/0008-5472.CAN-24-3854

Figure Lengend Snippet: Sequential activation of FADD converts anti–PD-1 responsiveness in ICI-resistant HCC mouse models. A, Schematic of sequential anti–PD-1 treatment and Fadd overexpression in orthotopic PD-1R tumor–bearing C57BL/6 mice. B, Kaplan–Meier survival analysis of mice from the indicated groups ( n = 8–10). C and D, Western blotting analysis of p-Fadd Ser191 and Fadd in tumors from control or AAV- Fadd –treated tumor-bearing mice ( C ) and CTNNB1 OE /MYC OE -induced spontaneous tumors ( D ). E, Schematic of sequential anti–PD-1 and ADT-OH treatment schedule in the CTNNB1 OE /MYC OE -induced spontaneous HCC model. F, Western blotting analysis of p-Fadd Ser191 and Fadd in tumors from control and ADT-OH–treated mice. G and H, Representative photos, hematoxylin and eosin staining ( G ), and enumeration of tumor nodules in the indicated groups on day 80 ( H ). Scale bar, 1,000 μm. I, Uniform Manifold Approximation and Projection (UMAP) plots showing high-dimensional flow cytometry analysis of tumor-infiltrating CD45 + leukocytes from the indicated groups of mice ( n = 6). UMAP plots consist of 12,000 cells each and are representative of concatenated samples within each group. Indicated immune cell clusters are highlighted in the indicated colors, and their proportions as percentages of CD45 + leukocytes are quantified ( J ). DC, dendritic cell; NA, no annotation. K, Heat map showing Pearson correlations among the number of tumor nodules; p-Fadd Ser191 ; Fadd; CCL5; the proportions of total, CCR5 + , IFNγ + , and TNFα + CD8 + T cells; TUNEL scores; and the concentrations of IFNγ and TNFα. L, Kaplan–Meier survival analysis of mice from the indicated groups ( n = 10–14); two-sided log-rank (Mantel–Cox) test. M, The FADD/CCL5/CD8a signature in patients with HCC treated with atezolizumab alone or in combination with bevacizumab (anti-VEGF). Data are presented as the mean ± SD for at least two (mouse models) or three (Western blotting) independent experiments and analyzed with the unpaired, two-tailed Student t test. Correlation analyses were performed using single-tailed Pearson correlations. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: Additionally, CCL5 nAbs (R&D Systems, #MAB678-SP) or rCCL5 (R&D Systems, #278-RN-050/CF) were injected into HepG2- FADD or Huh7-sh FADD subcutaneous tumors, followed by T-cell adoptive transfer and IHC staining as described above.

Techniques: Activation Assay, Over Expression, Western Blot, Control, Staining, Flow Cytometry, TUNEL Assay, Two Tailed Test