Journal: Frontiers in Immunology

Article Title: The Involvement of the Chemokine RANTES in Regulating Luminal Acidification in Rat Epididymis

doi: 10.3389/fimmu.2020.583274

Figure Lengend Snippet: Expression of RANTES receptors CCR1, CCR3 and CCR5 in the adult rat epididymis. (A) Expression of CCR1, CCR3 and CCR5 in rat epididymis were detected by RT -PCR. (B) Immunohistochemistry of CCR1 and CCR5 in rat epididymis. Black arrows: positive signals for CCR1 or CCR5. (C) Double IF staining was applied to detected the co-localization of RANTES with CCR1 and CCR5 in rat epididymis. The green signal of RANTES (Alexa 488 labeled) coincide with that of the red signal of CCR1 and CCR5 (Cy3 labeled). Bars = 20 μm.

Article Snippet: The same method was used in the experiment to detect the localization of CCR1 and CCR5 in rat epididymis, where the goat anti-CCR1 antibody and goat anti-CCR5 antibody (Santa Cruz Biotechnology) were used, with secondary antibodies, biotinylated anti-goat IgG (Sigma, St. Louis, MO, United States), applied for immunohistochemical staining.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Immunohistochemistry, Staining, Labeling

Journal: Frontiers in Immunology

Article Title: The Involvement of the Chemokine RANTES in Regulating Luminal Acidification in Rat Epididymis

doi: 10.3389/fimmu.2020.583274

Figure Lengend Snippet: Effects of rat vasectomy on the luminal pH and the expression of V-ATPase, RANTES, CCR1, CCR5, iNOS, and RAS. (A) 8 weeks after rat bilateral vasectomy, luminal fluid pH of cauda epididymis increased and remained alkaline. (B) The expression of V-ATPase in rat cauda epididymis was detected by Real time-PCR, WB and IF staining. (C) 8 weeks after rat bilateral vasectomy, the expression levels of RANTES, CCR1, CCR5, and iNOS mRNA and protein in cauda epididymis were decreased revealed by Real time-PCR and WB. (D) The tissue homogenate ANG II concentration were measured by RIA and the expression of AGTR2 was detected by Real time -PCR and WB. * P < 0.05, N = 5–6, Bar = 20 μm.

Article Snippet: The same method was used in the experiment to detect the localization of CCR1 and CCR5 in rat epididymis, where the goat anti-CCR1 antibody and goat anti-CCR5 antibody (Santa Cruz Biotechnology) were used, with secondary antibodies, biotinylated anti-goat IgG (Sigma, St. Louis, MO, United States), applied for immunohistochemical staining.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Staining, Concentration Assay

Journal: Frontiers in Immunology

Article Title: The Involvement of the Chemokine RANTES in Regulating Luminal Acidification in Rat Epididymis

doi: 10.3389/fimmu.2020.583274

Figure Lengend Snippet: Schematic representation of RANTES in the basal macrophages contributing to the intricate luminal acidic regulation in the epididymis. Firstly, epididymal luminal ANG II binds with its receptors AGTR2 expressed on the membrane of basal cells, inducing the expression of RANTES and its receptors, CCR1 and CCR5 in the basal macrophages. Secondly, RANTES could function with CCR1 and CCR5 to increase the expression of iNOs, which will lead to the up-regulation of NO. Finally, NO will diffuse to the adjacent clear cells to stimulate the expression of V-ATPase, causing the increased secretion of proton to the epididymal lumen to maintain the normal acidic microenvironment.

Article Snippet: The same method was used in the experiment to detect the localization of CCR1 and CCR5 in rat epididymis, where the goat anti-CCR1 antibody and goat anti-CCR5 antibody (Santa Cruz Biotechnology) were used, with secondary antibodies, biotinylated anti-goat IgG (Sigma, St. Louis, MO, United States), applied for immunohistochemical staining.

Techniques: Membrane, Expressing

Journal: Scandinavian journal of immunology

Article Title: A novel approach to regulate experimental visceral leishmaniasis in murine macrophages using CCR5 siRNA.

doi: 10.1111/j.1365-3083.2008.02076.x

Figure Lengend Snippet: Figure 1 Leishmania donovani induces CCR5 receptor expression in murine macrophages and J774A.1 cell line in a time-dependent manner. (A) L. donovani infection in BALB ⁄ c peritoneal macrophages for 0, 3, 6, 12, 24 and 48 h showed time-dependent increase in the CCR5 expres- sion up to 24 h and downregulation at further time points as shown in the immunoblot. J774A.1, a murine macrophage-like cell line, under identical condition showed a similar pattern of CCR5 expression. (B) Densitometric analysis of the blots.

Article Snippet: Goat anti-mouse CCR5 polyclonal antibody (Santa Cruz Biotechnology), anti-goat IgG and FITC-conjugated anti-goat IgG (Sigma), PE-conjugated CCR5 MoAb (e-Bioscience, San Diego, CA, USA) and Oligonucleotides (Sigma-Aldrich, St. Louis, MO, USA).

Techniques: Expressing, Infection, Western Blot

Journal: Scandinavian journal of immunology

Article Title: A novel approach to regulate experimental visceral leishmaniasis in murine macrophages using CCR5 siRNA.

doi: 10.1111/j.1365-3083.2008.02076.x

Figure Lengend Snippet: Figure 2 In vitro effect of CCR5 siRNA on the CCR5 expression in Leishmania donovani-infected macrophages. BALB ⁄ c peritoneal macrophages were transfected directly with 100 nm of CCR5 siRNA for 24 h and then infected with L. donovani for different time periods (3–24 h). (A) RT-PCR showed a decreased CCR5 mRNA expression due to CCR5 siRNA transfection. Lane 1 represented LD infection, lane 2: LD infection + CCR5siRNA and lane 3: LD infection + control siRNA. (B) Immunoblot showed significant downregulation of CCR5 protein expression (3–24 h). (C) Immunoflu- orescence studies demonstrating 40–60% silencing effect of CCR5siRNA on the surface expression of CCR5: (i) control; (ii) LD infection; (iii) LD + CCR5siRNA; (iv) LD + cont.siRNA. (D) FACS analysis confirmed successful silencing of CCR5 using siRNA transfection in primary macro- phages at 0, 3, 6 and 12 h of L. donovani infection. (E) Mean fluorescent intensity (MFI) was estimated in OD units and represented as bar diagram. Results were expressed as mean ± SD of data from three replicate experiments showing similar results (n = 3). Statistically significant differences (*P < 0.05) by one-way ANOVA.

Article Snippet: Goat anti-mouse CCR5 polyclonal antibody (Santa Cruz Biotechnology), anti-goat IgG and FITC-conjugated anti-goat IgG (Sigma), PE-conjugated CCR5 MoAb (e-Bioscience, San Diego, CA, USA) and Oligonucleotides (Sigma-Aldrich, St. Louis, MO, USA).

Techniques: In Vitro, Expressing, Infection, Transfection, Reverse Transcription Polymerase Chain Reaction, Control, Western Blot

Journal: Scandinavian journal of immunology

Article Title: A novel approach to regulate experimental visceral leishmaniasis in murine macrophages using CCR5 siRNA.

doi: 10.1111/j.1365-3083.2008.02076.x

Figure Lengend Snippet: Figure 3 Effect of CCR5 siRNA and CCR5 shRNA on Leishmania donovani entry and parasitic burden in murine macrophages and J774A.1 cell line. CCR5 siRNA and CCR5shRNA significantly reduced the parasitic burden in L. donovani-infected murine peritoneal macrophages and J774A.1 cell line. (A) Photographs of Giemsa-stained murine macrophages indicating the effect of CCR5 gene silencing by CCR5siRNA on the parasitic burden at 3, 6, 12 and 24 h. CCR5shRNA also showed similar results (pictures not shown). (B) The parasitic load was measured as the number of promastigotes attached per 100 macrophages after 3 h of infection and (C) number of amastigotes per 100 macrophages were counted after 12 h of infection in mur- ine peritoneal macrophages and J774A.1 cell line. (D) Number of amastigotes per 100 macrophages was counted after 24 h of infection in murine peritoneal macrophages and J774A.1 cell line. Results were expressed as mean ± SD of data from three replicate experiments showing similar results (n = 3). Statistically significant differences (*P < 0.05) by one-way ANOVA.

Article Snippet: Goat anti-mouse CCR5 polyclonal antibody (Santa Cruz Biotechnology), anti-goat IgG and FITC-conjugated anti-goat IgG (Sigma), PE-conjugated CCR5 MoAb (e-Bioscience, San Diego, CA, USA) and Oligonucleotides (Sigma-Aldrich, St. Louis, MO, USA).

Techniques: shRNA, Infection, Staining

Journal: Scandinavian journal of immunology

Article Title: A novel approach to regulate experimental visceral leishmaniasis in murine macrophages using CCR5 siRNA.

doi: 10.1111/j.1365-3083.2008.02076.x

Figure Lengend Snippet: Figure 5 Effect of CCR5siRNA and CCR5 shRNA on the proinflam- matory and anti-inflammatory cytokine balance in Leishmania donovani- infected murine macrophages and J774A.1 cell line. Sandwitch ELISA assay of proinflammatory cytokine TNF-a and anti-inflammatory cyto- kine IL-10 were carried out to measure the cytokine released in the cul- ture supernatant of murine macrophages and J774A.1 cells after 12 h incubation with L. donovani following CCR5siRNA.The ratio of TNF-a and IL-10 was calculated showing significant increase in CCR5 siRNA- transfected cells compared with the infection alone. However, the infected cells transfected with control siRNA or shRNA did not show any difference. Results were expressed as mean ± SD of data from three replicate experiments showing similar results (n = 3). Statistically significant differences (*P < 0.05) by one way ANOVA.

Article Snippet: Goat anti-mouse CCR5 polyclonal antibody (Santa Cruz Biotechnology), anti-goat IgG and FITC-conjugated anti-goat IgG (Sigma), PE-conjugated CCR5 MoAb (e-Bioscience, San Diego, CA, USA) and Oligonucleotides (Sigma-Aldrich, St. Louis, MO, USA).

Techniques: shRNA, Infection, Enzyme-linked Immunosorbent Assay, Incubation, Transfection, Control

Journal: Scandinavian journal of immunology

Article Title: A novel approach to regulate experimental visceral leishmaniasis in murine macrophages using CCR5 siRNA.

doi: 10.1111/j.1365-3083.2008.02076.x

Figure Lengend Snippet: Figure 4 Effect of CCR5siRNA and CCR5 shRNA on the nitric oxide generation in Leishmania donovani-infected macrophages after 48 h of infection. Nitric oxide generation was downregulated in murine macro- phages and J774A.1 cells after 48 h of infection, while transfection with CCR5siRNA and CCR5shRNA prior to infection showed significantly higher NO level. Infected cells which were transfected with control siR- NA ⁄ shRNA showed similar NO level as infection alone under identical condition. Nitric oxide released in the culture supernatant of LPS-stim- ulated L. donovani-infected macrophages was measured by Greiss reac- tion. Results were expressed as mean ± SD of data from three replicate experiments showing similar results (n = 3). Statistically significant differences (*P < 0.05) by one way ANOVA.

Article Snippet: Goat anti-mouse CCR5 polyclonal antibody (Santa Cruz Biotechnology), anti-goat IgG and FITC-conjugated anti-goat IgG (Sigma), PE-conjugated CCR5 MoAb (e-Bioscience, San Diego, CA, USA) and Oligonucleotides (Sigma-Aldrich, St. Louis, MO, USA).

Techniques: shRNA, Infection, Transfection, Control

Journal: PLoS ONE

Article Title: The enhancement of CCL2 and CCL5 by human bone marrow-derived mesenchymal stem/stromal cells might contribute to inflammatory suppression and axonal extension after spinal cord injury

doi: 10.1371/journal.pone.0230080

Figure Lengend Snippet: Primary antibodies used for immunohistochemistry.

Article Snippet: CCR5 , Rabbit , Bioss (Boston, MA) , bs-2514R , 500.

Techniques: Immunohistochemistry

Journal: Gene therapy

Article Title: Prevention of murine experimental autoimmune encephalomyelitis by in vivo expression of a novel recombinant immunotoxin DT390-RANTES.

doi: 10.1038/sj.gt.3302799

Figure Lengend Snippet: Figure 6 Immunohistochemical detection of CCR5+ cells in the CNS. (a) The perivascular infiltration in meninges of DT390-RANTES-treated mice; (b) the cerebral infiltration of DT390-RANTES-treated mice; (c) the perivascular infiltration in meninges of PBS-treated mice; (d) the cerebral infiltration of PBS-treated mice; (e) the perivascular infiltration in meninges of normal mice; and (f) the cerebral infiltration of normal mice ( 40).

Article Snippet: Sections were incubated with a goat anti-mouse CCR5 antibody (Boster Biotechnological Corp., Wuhan, Hubei, China) at 41C overnight.

Techniques: Immunohistochemical staining

Journal:

Article Title: Neurotrophins modulate the expression of chemokine receptors in the brain

doi: 10.1007/s13365-010-0004-3

Figure Lengend Snippet: Immunohistochemical analysis of CCR5 and CXCR4 immunoreactivity in mouse brain. a–d, sections were stained with CCR5 (red) and NeuN (green) antibodies. e–h, sections were stained with CXCR4 (green) and NeuN (red) antibodies as described in the Materials and Methods section. Orange denotes co-localization. a and e, cerebellar cortex; b and f, hippocampus; c and g, striatum; d and h, cerebral cortex. White arrows indicate neuronal cell bodies, and purple arrows indicate fibers. Images are higher magnifications (60×) of inserts. Bar = 20 µm; bar in inserts are 100 µm

Article Snippet: Floating sections were incubated overnight at 4°C in primary polyclonal antibodies (CXCR4, 1:200, Santa Cruz Biotech, Inc, Santa Cruz, CA; CCR5, 1:100, Abcam, San Francisco, CA) followed by biotinylated secondary antibodies for 1 h at room temperature.

Techniques: Immunohistochemical staining, Staining

Journal:

Article Title: Neurotrophins modulate the expression of chemokine receptors in the brain

doi: 10.1007/s13365-010-0004-3

Figure Lengend Snippet: NGF and NT-3 heterozygous mice exhibit higher levels of CXCR4 mRNA than wild type. The mRNA levels for CCR5 and CXCR4 were determined in 4-month-old C57BL/6 mice using qRT-PCR as described in the Materials and Methods section. a NGF het mice, b NT-3 het mice. Data were normalized to β-actin mRNA and expressed as arbitrary units (AU, mean±SEM of five independent samples). *p<0.05, **p<0.005 vs wild type using a one-way ANOVA and Student’s t-test

Article Snippet: Floating sections were incubated overnight at 4°C in primary polyclonal antibodies (CXCR4, 1:200, Santa Cruz Biotech, Inc, Santa Cruz, CA; CCR5, 1:100, Abcam, San Francisco, CA) followed by biotinylated secondary antibodies for 1 h at room temperature.

Techniques: Quantitative RT-PCR

Journal: PLoS ONE

Article Title: The Effects of the Recombinant CCR5 T4 Lysozyme Fusion Protein on HIV-1 Infection

doi: 10.1371/journal.pone.0131894

Figure Lengend Snippet: A) HIV-1 co-receptor function was estimated using cell-cell fusion assays. 3T3.T4 cells were transfected with 15 μg pSC-CCR5-T4L, then infected with vaccinia virus encoding LacZ gene under T7 promotor and used as target cells. The target cells were mixed with HeLa effector cells, expressing BaL Env protein and bacteriophage T7 RNA polymerase. After incubation for 2.5 h at 37°C, the amount of β-galactosidase activity was measured. WT-CCR5 was used as a positive control. B) CCR5 expression on the cell surface was confirmed using FACS. The level of CCR5 expression on above target 3T3.T4 cells was measured by flow cytometry using either phycoerythrin (PE)-conjugated 2D7 or phycoerythrin (PE)-conjugated 3A9 monoclonal antibodies. C and D) CCR5-T4L was more sensitive to CCR5 agonists maraviroc and TAK-779 inhibition. Different concentrations of maraviroc or TAK-779 were used to pretreat target 3T3.T4 cells transfected with pSC-CCR5-T4L or pSC-CCR5 for 1 h at 37°C. After washing with PBS, cell-cell fusion was performed. E and F) CCR5-T4L inhibited WT-CCR5 by reducing its expression on the cell surface. Different amounts of CCR5-T4L or WT-CCR5 cDNA (0 μg、 1 μg、 2.5 μg、5 μg、 7.5 μg and 10 μg) were co-transfected into 3T3.T4 cells. The effect of R5 HIV-1 Env-mediated cell-cell fusion was examined (E) and the level of CCR5 expression on the cell surface was measured by flow cytometry using a PE-conjugated monoclonal antibody (2D7) (F). G) The inhibition by CCR5-T4L decreased as the amounts of CCR5-T4L and WT-CCR5 increased. CCR5 expression was measured using flow cytometry and a PE-conjugated monoclonal antibody (2D7). The averaged mean fluorescence values (MFVs) for CCR5 from three experiments are plotted as a bar diagram, where WT-CCR5 expression after transfection with WT-CCR5 plus empty vector is set at 100. H and I) The interaction between CCR5-T4L and WT-CCR5 was tested using co-immunoprecipitation. 3T3.T4 cells co-transfected with CCR5-T4L and WT-CCR5 were lysed with RIPA buffer. Lysates were prepared and immunoprecipitated with the CCR5 C-terminal antibody, fractionated by SDS-PAGE, and immunoblotted. Blots were probed with the N-terminal CCR5 antibody (H), stripped, and reprobed with the anti-6×His antibody (I). Following the primary antibody reaction, blots were washed and probed with the anti-mouse antibody conjugated to HRP. Blots were exposed to X-ray film after reaction with the HRP substrate.

Article Snippet: Mouse monoclonal (sc-32304) and goat polyclonal (sc-6128) anti-CCR5 antibodies were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX).

Techniques: Transfection, Infection, Virus, Expressing, Incubation, Activity Assay, Positive Control, Flow Cytometry, Bioprocessing, Inhibition, Fluorescence, Plasmid Preparation, Immunoprecipitation, SDS Page

Journal: PLoS ONE

Article Title: The Effects of the Recombinant CCR5 T4 Lysozyme Fusion Protein on HIV-1 Infection

doi: 10.1371/journal.pone.0131894

Figure Lengend Snippet: A) Large scale purification and identification of recombinant CCR5-T4L. Recombinant CCR5-T4L was expressed in E . coli . The pET-20b expression vector was transformed into Rosetta 2 (DE3) golden BL21 pLysS cells and analyzed using Coomassie brilliant blue R-250. Lane M: protein marker; lane 1: uninduced bacterial lysate; lane 2: IPTG-induced bacteria lysate; lane 3: small amount of soluble fraction purified on a Ni-nitrilotriacetic acid (NTA) histidine-binding column; lane 4: small amount of membrane fraction purified using a Ni-nitrilotriacetic acid (NTA) histidine-binding column; lane 5: large amount of soluble fraction purified by fast protein liquid chromatography (FPLC) using an AKTA purifier; lane 6: large amount of membrane fraction purified by fast protein liquid chromatography (FPLC) using an AKTA purifier. B and C) Western blot analyses using the anti-6×His tag monoclonal or anti-human CCR5 monoclonal antibodies (3A9).

Article Snippet: Mouse monoclonal (sc-32304) and goat polyclonal (sc-6128) anti-CCR5 antibodies were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX).

Techniques: Purification, Recombinant, Expressing, Plasmid Preparation, Transformation Assay, Marker, Bacteria, Binding Assay, Membrane, Fast Protein Liquid Chromatography, Western Blot, Bioprocessing

Journal: PLoS ONE

Article Title: The Effects of the Recombinant CCR5 T4 Lysozyme Fusion Protein on HIV-1 Infection

doi: 10.1371/journal.pone.0131894

Figure Lengend Snippet: A and D) The inhibitory activities using cell-cell fusion assays. Effector HeLa cells co-expressing the R5 viral envelope BaL (A) or X4 (D) envelope LAV were pre-incubated for 30 min at 37°C with increasing concentrations of soluble recombinant CCR5-T4L plus 2 nM of soluble CD4. After washing with PBS, cell-cell fusion was performed. B and E) The inhibitory activities using single cycle viral infection assays in THP1 cells. Cells were pre-cultured overnight and infected with BaL or IIIB at 100 TCID 50 in the presence or absence of soluble recombinant CCR5-T4L. Luciferase activity was analyzed using a luciferase kit 8 days post-infection. C and F) The inhibitory activities using replication competent viral assays with BaL strain virus- (C) and IIIB strain virus- (F) in THP1 cells. CCR5-tropic HIV-1 ADA was used as a positive control. THP1 cells (1×10 6 cells per well) were pre-cultured overnight and infected with BaL or IIIB at 100 TCID 50 in the presence or absence of soluble recombinant CCR5-T4L overnight. Supernatants were collected 7 days post-infection and tested for p24 antigen using ELISA. Data are the average from three independent experiments. Recombinant ligand CCL5 and PBS were used as controls.

Article Snippet: Mouse monoclonal (sc-32304) and goat polyclonal (sc-6128) anti-CCR5 antibodies were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX).

Techniques: Expressing, Incubation, Recombinant, Infection, Cell Culture, Luciferase, Activity Assay, Virus, Positive Control, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: The Effects of the Recombinant CCR5 T4 Lysozyme Fusion Protein on HIV-1 Infection

doi: 10.1371/journal.pone.0131894

Figure Lengend Snippet: A and B) Effect of soluble recombinant CCR5-T4L or CCL5 on macrophages using cell-cell fusion assays (A) and single round virus infection assays (B). Macrophages cultured for 7 days were stimulated for 24 h at 37°C with CCR5-T4L (1 μg/ml) or CCL5 (1 μg/ml) prior to HIV-1 Env-mediated cell-cell fusion or single round virus infection. C, D, G, H, and I) Effect of soluble recombinant CCR5-T4L on HIV BaL infection in macrophages. Macrophages cultured for 7 days were stimulated with CCR5-T4L (1 μg/ml) for 24 h at 37°C prior to HIV-1 Bal infection. Cultured supernatant was collected 8 days post-infection, and cells were collected 12 days post-infection. Supernatants were subjected to RT assays (C), total RNA was evaluated for HIV-1 gag expression using real-time PCR (D), and total protein extracted from cells was evaluated for HIV-1 p24 protein expression by western blot analyses (G and H) and GAPDH (I). Representative blots from three independent experiments are shown. Densitometry analyses of the blot were performed using Image J 1.44 software (NIH) and plotted into graphs (n = 3). E and F) CCR5-T4L suppresses HIV-1 replication in macrophages. Macrophages were cultured at 37°C for 24 h in conditioned media in the presence or absence of CCR5-T4L (1 μg/ml) prior to HIV-1 infection or simultaneously or 8 h post-infection. Supernatants were collected 8 days post-infection, and cells were collected 12 days post-infection. Supernatants were subjected to RT assays (E) and total RNA was evaluated for HIV-1 gag expression using real-time PCR (F). Data are expressed as RNA levels relative to control. The results represent the mean ± SD of three independent experiments.

Article Snippet: Mouse monoclonal (sc-32304) and goat polyclonal (sc-6128) anti-CCR5 antibodies were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX).

Techniques: Recombinant, Virus, Infection, Cell Culture, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Software, Control

Journal: PLoS ONE

Article Title: The Effects of the Recombinant CCR5 T4 Lysozyme Fusion Protein on HIV-1 Infection

doi: 10.1371/journal.pone.0131894

Figure Lengend Snippet: Anti-HIV activity of the soluble recombinant CCR5-T4L protein in PBMC assays.

Article Snippet: Mouse monoclonal (sc-32304) and goat polyclonal (sc-6128) anti-CCR5 antibodies were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX).

Techniques: Activity Assay, Recombinant

Journal: PLoS ONE

Article Title: The Effects of the Recombinant CCR5 T4 Lysozyme Fusion Protein on HIV-1 Infection

doi: 10.1371/journal.pone.0131894

Figure Lengend Snippet: Anti-HIV activity of the soluble recombinant CCR5-T4L protein in PBMCs from four different donors.

Article Snippet: Mouse monoclonal (sc-32304) and goat polyclonal (sc-6128) anti-CCR5 antibodies were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX).

Techniques: Activity Assay, Recombinant

Journal: PLoS ONE

Article Title: The Effects of the Recombinant CCR5 T4 Lysozyme Fusion Protein on HIV-1 Infection

doi: 10.1371/journal.pone.0131894

Figure Lengend Snippet: A and B) Effect of soluble recombinant CCR5-T4L or CCL5 on surface CCR5 expression in MDMs. MDMs were treated with soluble recombinant CCR5-T4L (1 μg/ml), CCL5 (1 μg/ml), or PBS for 24 h at 37°C. Surface (A) and intracellular CCR5 (B) were analyzed using flow cytometry and the PE-conjugated monoclonal antibody (2D7). Recombinant CCL5 protein was used as a control. The cellular distribution of CCR5 receptors was analyzed by fixing and permeabilizing cells using BD Cytofix/Cytoperm buffer. Data shown are from one representative experiment that was independently repeated at least three times. C) Dose-dependent effects of CCR5-T4L or CCL5 on surface CCR5 expression in MDM. D and E) Dose-dependent effects on MDM migration by CCR5-T4L (D) or CCL5 (100 nM) plus different concentrations of CCR5-T4L protein (E). F and G) Dose-dependent effects on [125I]-CCR5 binding by CCR5-T4L (F) or CCL5 (100 nM) plus different concentrations of CCR5-T4L protein (G). H and I) Dose-dependent effects on CCL5-induced [ 35 S]GTPγS binding to membranes from human macrophages cells treated with CCR5-T4L (H) or CCL5 (100 nM) plus different concentrations of CCR5-T4L protein (I). Data are the mean ± SD of triplicate cultures, which are representative of three experiments.

Article Snippet: Mouse monoclonal (sc-32304) and goat polyclonal (sc-6128) anti-CCR5 antibodies were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX).

Techniques: Recombinant, Expressing, Flow Cytometry, Control, Migration, Binding Assay