goat polyclonal  (R&D Systems)

Journal: Frontiers in Cell and Developmental Biology

Article Title: Malaria Parasites Hijack Host Receptors From Exosomes to Capture Lipoproteins

doi: 10.3389/fcell.2021.749153

Figure Lengend Snippet: HDL-uptake by Plasmodium -infected erythrocytes. (A) Uptake of HDL components by P. b erghei-pRBCs. Phospholipid and ester components were concomitantly observed around parasite, but HDL was not taken up by the nRBCs (arrows). Scale bar: 5 μm. (B) Pb -pRBCs western blotted for Apo A-I, using a polyclonal primary Ab reactive to both mouse and human Apo A-I. The membrane fraction showed Apo A-I positivity with or without the addition of human HDL. (C) Flow cytometric analysis of DiO-labelled HDL uptake. Synchronized ring-stage P. falciparum incubated for 24 h with 500 μg/ml of HDL. HDL uptake was competitively inhibited by unlabeled HDL. (D) Chemical structure of BLT-1. (E) BLT-1 dose-dependent inhibition of DiO-labelled HDL uptake to Pb -pRBCs. The numbers in panels represent fraction (%) of DiO-positive cells out of total cells.

Article Snippet: Exosomes from 50 μl of equally pooled plasma from five mice per group were sequentially incubated at room temperature for 1 h with goat polyclonal anti-CD36 Ab (AF2519, R and D Systems) and HRP-labelled bovine anti-goat IgG heavy and light chains, known to be minimally cross-reactive with mouse and rat serum proteins (805–035-180, Jackson ImmunoResearch, West Grove, PA, United States).

Techniques: Infection, Western Blot, Membrane, Incubation, Inhibition

Journal: Frontiers in Cell and Developmental Biology

Article Title: Malaria Parasites Hijack Host Receptors From Exosomes to Capture Lipoproteins

doi: 10.3389/fcell.2021.749153

Figure Lengend Snippet: Immunocytochemical analysis of Pb -pRBCs for scavenger receptors and CD41. (A) Immunocytochemical analysis of Pb -pRBCs. The CD36 signals (pink) were weak in the pRBCs on days 5 and 8 (ring stage), but were abundantly detected on days 11 and 14 (schizonts). On day 11, CD36 appeared as a spot corresponding to each nucleus in the schizont’s body. SR-B1, which was detected in the pRBC membrane, was negligible in the internal parasites (arrow). Scale bar: 5 μm (B) Immunocytochemical analysis of CD36 in BM-transplanted mouse erythrocytes 6 days after Pb infection. CD36 (pink) was detected in pRBCs from the Kusabira-Orange mouse, but not in the BM-transplanted or CD36 −/− mouse. Scale bar: 5 μm. (C) Immunocytochemical analysis of pRBCs. CD41 (pink) signals were absent on days 5 and 8 (ring stage), but a small signal emanated from the internal parasites on day 8. CD41-specific fluorescence became abundant on days 11 and 14 (schizont stage). On day 11, CD41 appeared as spots corresponding to nuclei in the schizont’s body, as was also seen with CD36. Scale bar: 5 μm. (D) Co-localization of CD36 (red) and CD41 (green). Scale bar: 5 μm.

Article Snippet: Exosomes from 50 μl of equally pooled plasma from five mice per group were sequentially incubated at room temperature for 1 h with goat polyclonal anti-CD36 Ab (AF2519, R and D Systems) and HRP-labelled bovine anti-goat IgG heavy and light chains, known to be minimally cross-reactive with mouse and rat serum proteins (805–035-180, Jackson ImmunoResearch, West Grove, PA, United States).

Techniques: Membrane, Infection, Fluorescence

Journal: Frontiers in Cell and Developmental Biology

Article Title: Malaria Parasites Hijack Host Receptors From Exosomes to Capture Lipoproteins

doi: 10.3389/fcell.2021.749153

Figure Lengend Snippet: Analyses of the exosomes isolated from Pb -parasitized mouse plasma or from cultured cells differentiated from CMK11-5 cells. (A) Electron micrograph of the plasma exosomes (arrows) on day 8 post-infection. Scale bar: 333 nm. (B) Western blot of exosomes from plasma, platelets, and pRBCs. CD36, CD41 and an exosome marker (ALIX) were detected on days 5 and 8 post-infection. (C) Detection of exosomes doubly-positive for CD36 and CD41 by sandwich ELISA (duplicated). Each group of exosomes was isolated from the ‘pooled plasma’ of five mice. PBS and exosomes from a CD36 −/− mouse were used as negative controls. On days 5 and 8 post-infection, increased OD450 nm was observed. (D) immunoblot analysis for CD36 and CD41 in cultured cells differentiated from CMK11-5 cells or the exosomes released from those cells. (E) Immunocytochemical analysis of erythrocytes from a Pb -parasitized CD36 −/− mouse 6 h after adding CMK11-5-derived exosomes. CD36 and CD41 were detected from internal Pb (pink). NC: parasitized erythrocytes incubated with CMK11-5-derived exosomes and immunostained without primary Abs but with secondary Abs. Scale bar: 5 μm. (F) Trafficking of DiI-labelled exosomes derived from CMK11-5 cells into erythrocytes 1 h after exosome addition. DiI was observed in pRBCs (red) but not in uninfected ones. Scale bar: 5 μm.

Article Snippet: Exosomes from 50 μl of equally pooled plasma from five mice per group were sequentially incubated at room temperature for 1 h with goat polyclonal anti-CD36 Ab (AF2519, R and D Systems) and HRP-labelled bovine anti-goat IgG heavy and light chains, known to be minimally cross-reactive with mouse and rat serum proteins (805–035-180, Jackson ImmunoResearch, West Grove, PA, United States).

Techniques: Isolation, Clinical Proteomics, Cell Culture, Infection, Western Blot, Marker, Sandwich ELISA, Derivative Assay, Incubation

Journal: Frontiers in Cell and Developmental Biology

Article Title: Malaria Parasites Hijack Host Receptors From Exosomes to Capture Lipoproteins

doi: 10.3389/fcell.2021.749153

Figure Lengend Snippet: In vivo Pb survival and growth (parasitemia). (A) Kaplan-Meier survival curve of Pb -infected mice. CD36 −/− mice survived for significantly longer than the C57BL6 mice. (B) Parasitemia during Pb infection.

Article Snippet: Exosomes from 50 μl of equally pooled plasma from five mice per group were sequentially incubated at room temperature for 1 h with goat polyclonal anti-CD36 Ab (AF2519, R and D Systems) and HRP-labelled bovine anti-goat IgG heavy and light chains, known to be minimally cross-reactive with mouse and rat serum proteins (805–035-180, Jackson ImmunoResearch, West Grove, PA, United States).

Techniques: In Vivo, Infection

Journal: Frontiers in Cell and Developmental Biology

Article Title: Malaria Parasites Hijack Host Receptors From Exosomes to Capture Lipoproteins

doi: 10.3389/fcell.2021.749153

Figure Lengend Snippet: Proposed mechanism of exosome-based CD36 delivery and HDL capture in pRBCs. Pf EMP1 in P. falciparum and proteins that have similar binding ability to CD36 in mice malaria catch exosomes to hijack the receptors.

Article Snippet: Exosomes from 50 μl of equally pooled plasma from five mice per group were sequentially incubated at room temperature for 1 h with goat polyclonal anti-CD36 Ab (AF2519, R and D Systems) and HRP-labelled bovine anti-goat IgG heavy and light chains, known to be minimally cross-reactive with mouse and rat serum proteins (805–035-180, Jackson ImmunoResearch, West Grove, PA, United States).

Techniques: Binding Assay

Journal: Biomedical research (Tokyo, Japan)

Article Title: A novel role for scavenger receptor B1 as a contributor to the capture of specific volatile odorants in the nasal cavity.

doi: 10.2220/biomedres.39.117

Figure Lengend Snippet: Fig. 5 RT-PCR and western immunoblot analyses for assessment of SR-B1 expression in the nasal tissue of mice. (A) RT- PCR analysis. A representative result for RT-PCR detection of SR-B1 mRNA from a nasal sample is presented in the left panel (lane 1). A DNA size marker (100 bp DNA Ladder, New England Biolabs Japan, Tokyo) was loaded in lane M. The positions at which 100-, 500-, and 1000-bp fragments migrate are indicated on the left in base pairs (bp). Right panels show representative data for RT-PCR detection of SR-B1 (upper) and β-actin (lower) mRNAs in the nasal mucosa (Nasal) and liver of mice. (B) Western immunoblot analysis. Lysates obtained from the nasal mucosa (Nasal) and liver of mice were analysed by SDS-PAGE and western blotting with an anti-SR-B1 antibody (NB400-104) (left panel). In the right panel, a western blot with an anti-CD36 antibody (AF2519) for nasal mucosa and liver samples is shown. Samples were analysed in duplicate for either of the receptors. In either panel, the positions where marker proteins migrate are indicated on the left in kilo Daltons (kDa).

Article Snippet: For probing CD36, a goat polyclonal anti-CD36 antibody (1 : 2000, AF2519; R&D Systems, Minnesota, MN, USA) and peroxidase-labelled polyclonal rabbit anti-goat IgG secondary antibody (1 : 1000, DAKO P0449; Agilent) were used as primary and secondary antibodies, respectively.

Techniques: Reverse Transcription Polymerase Chain Reaction, Western Blot, Expressing, Marker, SDS Page

Journal: Biomedical research (Tokyo, Japan)

Article Title: A novel role for scavenger receptor B1 as a contributor to the capture of specific volatile odorants in the nasal cavity.

doi: 10.2220/biomedres.39.117

Figure Lengend Snippet: Fig. 7 Double immunostaining of the olfactory epithelium in mice with an anti-CD36 antibody (AF2519) (top panel) and an anti-SR-B1 antibody (TA301489) (middle panel). The merged image is shown in the bottom panel. Within the image, ten or more CD36-immunopositive olfactory sen- sory neurons in the deeper epithelial layer are illustrated (red). Of these, the ones in which the slender process be- ing extended to the apical surface (one of the histologic signatures of olfactory sensory neurons) is identifiable, are indicated by arrowheads. Bar in the top panel: 20 μm.

Article Snippet: For probing CD36, a goat polyclonal anti-CD36 antibody (1 : 2000, AF2519; R&D Systems, Minnesota, MN, USA) and peroxidase-labelled polyclonal rabbit anti-goat IgG secondary antibody (1 : 1000, DAKO P0449; Agilent) were used as primary and secondary antibodies, respectively.

Techniques: Double Immunostaining

Journal: PLoS ONE

Article Title: CD36 Differently Regulates Macrophage Responses to Smooth and Rough Lipopolysaccharide

doi: 10.1371/journal.pone.0153558

Figure Lengend Snippet: (A) bS-LPS binds dose-dependently to WT PEMs. (B) WT, CD36-/- and SR-A-/- PEMs exhibit similar binding of bS-LPS, which is fully blocked by anti-CD14 mAb. (C) Binding of bS-LPS at 37°C to metabolically poisoned PEMs is inhibited by anti-CD14 mAb and by an excess of unlabeled S-LPS. Graphs show averages +SEM from 3 independent experiments (B) or results of single experiments, performed in 4 replicates, each representative of 3 such experiments performed (A, C). *, significant inhibition of bS-LPS binding (p<0.05), according to Student’s t-test (B) or ANOVA, followed by the Dunnett’s post-test (C); A.U., arbitrary units.

Article Snippet: Bound rCD36 was detected by 1-h incubation with 2 μg/ml of polyclonal goat anti-mouse CD36 Ab (R&D Systems), followed by 1-h incubation with 4 μg/ml of HRP-conjugated F(ab’) 2 fragments of donkey anti-goat IgG Ab (Rockland) in BSA-PBS.

Techniques: Binding Assay, Metabolic Labelling, Inhibition

Journal: PLoS ONE

Article Title: CD36 Differently Regulates Macrophage Responses to Smooth and Rough Lipopolysaccharide

doi: 10.1371/journal.pone.0153558

Figure Lengend Snippet: (A) Incubation with 5 μg/ml rCD14, but not with rCD36 partially removes bS-LPS from rLBP. (B) rLBP, but not rCD36 or serum depletes rCD14 of bound bS-LPS. (C) rLBP binds very weakly to plate-adsorbed rCD14, but not to rCD36 and this binding is only slightly increased by 1 μg/ml S-LPS. (D) bS-LPS binds much more strongly to plate-adsorbed rCD36 in BSA-PBS than in FCS-PBS. (E) rCD36 binding to plate-adsorbed GA-BSA is inhibited by 0.2 mg/ml LTA, 0.1 mg/ml DS and anti-CD36 mAb, whereas 0.2 mg/ml S-LPS inhibits rCD36 binding only in the absence of serum. Chondroitin sulfate (CS) and control IgA have no effect on the binding. (F) A large portion of bS-LPS bound to rCD36 is removed by serum component(s) distinct from CD14 or LBP. (G) Relative to WT controls, internalization of bS-LPS/pHr-SAV complexes is significantly decreased in CD36-/-, but not SR-A-/- PEMs. Graphs show means ± SEM of 3–5 replicates, obtained in single experiments, each representative of at least 3 similar experiments performed (A-F) or averages + SEM from 4 independent experiments (G). Data were analyzed with the regular (A, B, E and F) or repeated measures (G) ANOVA, followed by the Dunnett’s post-test. *, p < 0.05; OD, optical density.

Article Snippet: Bound rCD36 was detected by 1-h incubation with 2 μg/ml of polyclonal goat anti-mouse CD36 Ab (R&D Systems), followed by 1-h incubation with 4 μg/ml of HRP-conjugated F(ab’) 2 fragments of donkey anti-goat IgG Ab (Rockland) in BSA-PBS.

Techniques: Incubation, Binding Assay, Control

Journal: PLoS ONE

Article Title: CD36 Differently Regulates Macrophage Responses to Smooth and Rough Lipopolysaccharide

doi: 10.1371/journal.pone.0153558

Figure Lengend Snippet: (A) S-LPS at 1 μg/ml enhances binding of rCD36 to plate-adsorbed rCD14. (B) CD36-/- PEMs exhibit neither basal nor 1 μg/ml S-LPS-stimulated rCD14 binding. (C) S-LPS-stimulated binding of rCD14 to PEMs is blocked by anti-CD36 mAb, but not by anti-TLR2 or anti-TLR4/MD-2 mAb. (D) During 40 min co-incubation at 37°C, 200 ng/ml S-LPS stimulates much stronger rCD14 binding to PEMs than the same concentration of R-LPS. Graphs show means ± SEM of 3–4 replicates, obtained in single experiments, each representative of at least 3 similar experiments performed. Data on graphs C and D were analyzed with ANOVA, followed by the Dunnett’s post-test. *, p < 0.05; HRP-anti-Fc, HRP-conjugated F(ab’) 2 fragments of goat antibodies specific for Fc fragments of human IgG.

Article Snippet: Bound rCD36 was detected by 1-h incubation with 2 μg/ml of polyclonal goat anti-mouse CD36 Ab (R&D Systems), followed by 1-h incubation with 4 μg/ml of HRP-conjugated F(ab’) 2 fragments of donkey anti-goat IgG Ab (Rockland) in BSA-PBS.

Techniques: Binding Assay, Incubation, Concentration Assay

Journal: PLoS ONE

Article Title: CD36 Differently Regulates Macrophage Responses to Smooth and Rough Lipopolysaccharide

doi: 10.1371/journal.pone.0153558

Figure Lengend Snippet: Effects of CD36 or SR-A deficiency and of anti-CD14 mAb on TNF-α and RANTES production, stimulated by 1-h incubation on ice with 1 μ g/ml S-LPS (A-B) or R-LPS (C-D). (A) Anti-CD14 mAb blocks both TNF-α and RANTES production, stimulated by S-LPS. (B) S-LPS stimulates significantly higher cytokine production in CD36-/- than in WT or SR-A-/- PEMs. (C) R-LPS-stimulated cytokine production is more strongly inhibited by anti-CD14 mAb in CD36-/- than in WT or SR-A-/- PEMs. (D) R-LPS stimulates similar cytokine production in WT, CD36-/- and SR-A-/- PEMs. Graphs show means +SEM from 4–6 independent experiments, each performed in 4 replicates. Data on graphs A and C were analyzed with one-sample t-test, and on graphs B and D with ANOVA, followed by the Dunnett’s test. *, p < 0.05.

Article Snippet: Bound rCD36 was detected by 1-h incubation with 2 μg/ml of polyclonal goat anti-mouse CD36 Ab (R&D Systems), followed by 1-h incubation with 4 μg/ml of HRP-conjugated F(ab’) 2 fragments of donkey anti-goat IgG Ab (Rockland) in BSA-PBS.

Techniques: Incubation

Journal: PLoS ONE

Article Title: CD36 Differently Regulates Macrophage Responses to Smooth and Rough Lipopolysaccharide

doi: 10.1371/journal.pone.0153558

Figure Lengend Snippet: (A) Binding of mAbs to PEMs pre-incubated with the indicated concentrations of S-LPS. (B) Forty-min pre-incubation at 37°C with 200 ng/ml S-LPS, but not with R-LPS in FCS-RPMI more strongly inhibits MTS510 mAb binding to TLR4/MD-2 on CD36-/- than on WT PEMs. In contrast, in BSA-RPMI both S-LPS and R-LPS produce significant inhibition of MTS510 mAb binding only in WT PEMs. (C) Forty-min stimulation in FCS-RPMI with 200 ng/ml S-LPS, but not with R-LPS induces significantly higher TNF-α production in WT than in CD36-/- PEMs (left panel). In contrast, R-LPS, but not S-LPS stimulates significantly lower RANTES production in CD36-/- PEMs (right panel). In BSA-RPMI, CD36-/- PEMs exhibit severe impairment of cytokine production in response to both S-LPS and R-LPS. (D) In both WT and CD36-/- PEMs cytokine production stimulated by 40-min incubation with 200 ng/ml S-LPS in serum-free medium is blocked completely by anti-CD14 mAb. Anti-CD14 mAb also blocks cytokine production stimulated by R-LPS in CD36-/- PEMs, whereas it exerts only partial inhibition in WT PEMs. Data shown on graphs A and D are means +SEM of triplicates, obtained in single experiments, which were performed 2 (A) or 3 (D) times with similar results. Graphs B-C show means +SEM from 4–6 independent experiments, each performed in 4 replicates. *, significant inhibition or difference between WT and CD36-/- PEMs (p < 0.05 in Student’s t-test).

Article Snippet: Bound rCD36 was detected by 1-h incubation with 2 μg/ml of polyclonal goat anti-mouse CD36 Ab (R&D Systems), followed by 1-h incubation with 4 μg/ml of HRP-conjugated F(ab’) 2 fragments of donkey anti-goat IgG Ab (Rockland) in BSA-PBS.

Techniques: Binding Assay, Incubation, Inhibition

Journal: PLoS ONE

Article Title: CD36 Differently Regulates Macrophage Responses to Smooth and Rough Lipopolysaccharide

doi: 10.1371/journal.pone.0153558

Figure Lengend Snippet: (A) Hydroxy-dynasore (Dyn-OH) at 30 μM blocks RANTES, but enhances TNF-α production, stimulated by 1-h incubation on ice with 1 μg/ml LPS. Cytochalasin D (Cyt-D) at 10 μM has no effect on RANTES, but inhibits TNF-α production. (B, C) One μg/ml S-LPS (B) and R-LPS (C) produce stronger down-regulation of cell surface TLR4/MD-2 expression in WT than in CD36-/- PEMs. (D) Internalization of CD14-bound bS-LPS/pHr-SAV complexes into acidic endosomes is blocked by both Dyn-OH and Cyt-D. A JNK inhibitor (JNKi) at 20 μM produces stronger inhibition of the internalization in WT than in CD36-/- PEMs. (E) Seventy-min uptake of 10 μg/ml BO-LPS by WT PEMs is inhibited by Cyt-D, but enhanced by Dyn-OH. Graphs show means +SEM from 3–4 independent experiments, each performed in 3–4 replicates. *, significant inhibition (significantly different (p < 0.05) from 100 in one-sample t-test) (A), significant difference between WT and CD36-/- PEMs (p < 0.05 in two-tailed t-test) (B, C, D), or significantly different from the solvent control (p < 0.05 in ANOVA, followed by the Dunnett’s post-test) (E).

Article Snippet: Bound rCD36 was detected by 1-h incubation with 2 μg/ml of polyclonal goat anti-mouse CD36 Ab (R&D Systems), followed by 1-h incubation with 4 μg/ml of HRP-conjugated F(ab’) 2 fragments of donkey anti-goat IgG Ab (Rockland) in BSA-PBS.

Techniques: Incubation, Expressing, Inhibition, Two Tailed Test, Solvent, Control