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: Aging (Albany NY)

Article Title: Tanshinone IIA attenuates atherosclerosis via inhibiting NLRP3 inflammasome activation

doi: 10.18632/aging.202202

Figure Lengend Snippet: Effects of the LOX-1, CD36 and SR-A1 siRNAs on oxLDL-induced IL-1β productions and effects of oxLDL and Tan IIA on LOX-1 expression and LOX-1-mediated caspase-1activation/IL-1β release. ( A – C ) Effects of lentivirus-mediated LOX-1, CD36 and SR-A1 siRNAs (MOI = 30) on oxLDL-induced IL-1β productions in mouse macrophages measured by ELISA. ( D , E ) Effects of oxLDL on LOX-1 expression in macrophages measured by qRT-PCR and Western blot, respectively. ( F , G ) Effects of Tan IIA on oxLDL-induced LOX-1 expression determined by qRT-PCR and Western blot, respectively. ( H ) Effects of Tan IIA on oxLDL-induced LOX-1-mediated IL-1β expression in macrophages. ( I ) Effects of Tan IIA on oxLDL-induced LOX-1-mediated caspase-1 activation in macrophages.

Article Snippet: Tanshinone IIA, CA-074-Me (cathepsin B inhibitor) and the antibodies used for immunoblotting, including rabbit anti-mouse caspase-1 p10 (M-20), rabbit anti-mouse cathepsin B (FL-339) / goat anti-rabbit IgG-AP, Armenian hamster anti-mouse IL-1β (B122) / goat anti-Armenian hamster IgG-AP, goat anti-mouse or human GAPDH (L-18) / rabbit anti-goat IgG-AP, goat anti-mouse LOX-1 polyclonal antibody, goat anti-mouse P62 polyclonal antibody, goat anti-mouse CD36 polyclonal antibody (N-15), goat anti-mouse NLRP3 antibody (M-12), donkey anti-goat IgG-HRP, donkey anti-goat IgG-FITC were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Western Blot, Activation Assay

Journal: Aging (Albany NY)

Article Title: Tanshinone IIA attenuates atherosclerosis via inhibiting NLRP3 inflammasome activation

doi: 10.18632/aging.202202

Figure Lengend Snippet: Tan IIA inhibits oxLDL-induced CD36 expression by inhibiting Nrf2 expression and PPARγ activity. ( A , B ) Effects of oxLDL on CD36 expressions at mRNA levels measured by qRT-PCR and protein levels detected by Western blot in macrophage, respectively; GAPDH, glyceraldehyde-3-phosphate dehydrogenase as an internal control. ( C , D ) Effects of Tan IIA on oxLDL-induced CD36 expressions at mRNA levels and protein levels in macrophages, respectively. ( E ) Effects of Tan IIA on oxLDL-induced caspase-1 activation in the cells detected by Western blot. ( F ) Effects of Tan IIA on oxLDL-induced IL-1β productions measured by ELISA in macrophages. ( G , H ) Effects of Nrf2 and PPARγ gene knockdown by siRNA on CD36 expression on the cell membrane, respectively, detected by flow cytometry, presented by mean fluorescence intensity (MFI). ( I , J ) Effects of Tan IIA on oxLDL-mediated Nrf2 expression measured by RT-PCR and oxLDL-mediated PPARγ activity measured by a commercial kit in the cells.

Article Snippet: Tanshinone IIA, CA-074-Me (cathepsin B inhibitor) and the antibodies used for immunoblotting, including rabbit anti-mouse caspase-1 p10 (M-20), rabbit anti-mouse cathepsin B (FL-339) / goat anti-rabbit IgG-AP, Armenian hamster anti-mouse IL-1β (B122) / goat anti-Armenian hamster IgG-AP, goat anti-mouse or human GAPDH (L-18) / rabbit anti-goat IgG-AP, goat anti-mouse LOX-1 polyclonal antibody, goat anti-mouse P62 polyclonal antibody, goat anti-mouse CD36 polyclonal antibody (N-15), goat anti-mouse NLRP3 antibody (M-12), donkey anti-goat IgG-HRP, donkey anti-goat IgG-FITC were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Expressing, Activity Assay, Quantitative RT-PCR, Western Blot, Control, Activation Assay, Enzyme-linked Immunosorbent Assay, Knockdown, Membrane, Flow Cytometry, Fluorescence, Reverse Transcription Polymerase Chain Reaction

Journal: Aging (Albany NY)

Article Title: Tanshinone IIA attenuates atherosclerosis via inhibiting NLRP3 inflammasome activation

doi: 10.18632/aging.202202

Figure Lengend Snippet: Tan IIA inhibits oxLDL-induced macrophage lysosomal damage and cathepsin B release. ( A ) Fluorescence confocal assay for oxLDL-induced cathepsin B release from lysosome of mouse B6 macrophages or cathepsin B -/- B6 macrophages under the different treatments, respectively. The cells were treated with oxLDL (50 μg/mL) alone (2), oxLDL plus 2.5 μg/mL Tan IIA (3), oxLDL plus 10 μg/mL Tan IIA (4) and oxLDL plus Cathepsin B inhibitor CA-074-Me (5). The Cathepsin B -/- mouse macrophages (6) were treated with oxLDL alone. The small interfering RNA-mediated CD36 silenced-mouse B6 macrophages (7) were treated with oxLDL alone. All the cells were stained with Alexa 647-conjugated cholera toxin B (membrane staining, red), and then with DQ ovalbumin (cathepsin B staining, green) and Hoechst dye (nuclei staining, blue) after saponin treatment. ( B ) Western blot detection for Cathepsin B releases from cytosol of oxLDL-induced B6 macrophages or cathepsin B -/- B6 macrophages treated with digitonin and phenylmethylsulfonyl fluoride (PMSF). ( C ) ELISA assay for oxLDL-induced Il-1β releases from mouse B6 macrophages or cathepsin B -/- B6 macrophages under the different treatments.

Article Snippet: Tanshinone IIA, CA-074-Me (cathepsin B inhibitor) and the antibodies used for immunoblotting, including rabbit anti-mouse caspase-1 p10 (M-20), rabbit anti-mouse cathepsin B (FL-339) / goat anti-rabbit IgG-AP, Armenian hamster anti-mouse IL-1β (B122) / goat anti-Armenian hamster IgG-AP, goat anti-mouse or human GAPDH (L-18) / rabbit anti-goat IgG-AP, goat anti-mouse LOX-1 polyclonal antibody, goat anti-mouse P62 polyclonal antibody, goat anti-mouse CD36 polyclonal antibody (N-15), goat anti-mouse NLRP3 antibody (M-12), donkey anti-goat IgG-HRP, donkey anti-goat IgG-FITC were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Fluorescence, Confocal Assay, Small Interfering RNA, Staining, Membrane, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: Transcriptional Regulation of Thrombospondins and Its Functional Validation through CRISPR/Cas9 Mediated Gene Editing in Corpus Luteum of Water Buffalo (Bubalus Bubalis).

doi: 10.33594/000000038

Figure Lengend Snippet: Fig. 2. Demonstration of (A) representative blots of TSP1, TSP2, CD36 and GAPDH by immunoblotting at different stages of CL development in the riverine buffalo. The relative molecular weight of each protein is shown along the right side of each blot. The luteal proteins were loaded @ 100 mg/ well and resolved in 10% SDS-PAGE followed by electrotransfer to a PVDF membrane. Protein specific antibodies were used @ 1:500 while secondary antibody @1:2000 dilutions. GAPDH was used as reference protein. Relative expression of TSP and its receptors was analysed by densitometry using image J software (n = 6/group). One-way ANOVA to determine if treatment groups were significantly different. Tukey HSD test was done to find the pair-wise mean differences. Each bar represents Mean ± SEM. Different superscripts denote statistical significance (p<0.05). Abbreviations: CL, Corpus Luteum; TSP, Thrombospondin; CD, Cluster of differentiation.

Article Snippet: Antibodies and Growth factor Immunoblotting and immunohistochemistry were performed using goat polyclonal GAPDH (sc-48166; Santa Cruz Biotechnology, Inc., Dallas, TX), mouse monoclonal TSP1 (sc-393504; Santa Cruz Biotechnology, Inc., Dallas, TX), goat polyclonal TSP2 (sc-12313; Santa Cruz Biotechnology, Inc., Dallas, TX), goat polyclonal CD36 (sc-5522; Santa Cruz Biotechnology, Inc., Dallas, TX), mouse anti-goat IgG-HRP (sc-2354; Santa Cruz Biotechnology, Inc., Dallas, TX), goat anti-mouse IgG-HRP (sc-2005; Santa Cruz Biotechnology, Inc., Dallas, TX) bovine anti-goat IgG-CFL-647 (sc-362284, Lot#B0312) and goat anti-mouse IgG-CFL-647 (sc362257; Santa Cruz Biotechnology, Inc.).

Techniques: Western Blot, Molecular Weight, SDS Page, Electrotransfer, Membrane, Expressing, Software

Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: Transcriptional Regulation of Thrombospondins and Its Functional Validation through CRISPR/Cas9 Mediated Gene Editing in Corpus Luteum of Water Buffalo (Bubalus Bubalis).

doi: 10.33594/000000038

Figure Lengend Snippet: Fig. 5. Immunohistochemical localization of CD36 in the late CL stage of riverine buffalo. The 5 μm thick sections of CL were deparaffinised and rehydrated, followed by antigen retrieval. Primary TSP1 antibody was used at 1:200 while the FITC was used at 1:500. DAPI was used to counterstain nucleus. Fluorescent signals were captured by microscopy (Carl Zeiss Micro Imaging GmbH). Representative images from (A) bright field, (B) through (C) indicate intense immunoreactivity in late stages of CL which was localized predominantly in the cytoplasm of luteal cells. No primary antibody was used in the negative control (D). Scale bar = 50 μm. Abbreviations: LL, Large luteal cell; SL, Small luteal cell; FITC, Fluorescein isothiocyanate; DAPI, 40, 6-diamidino-2-phenylindole dihydrochloride.

Article Snippet: Antibodies and Growth factor Immunoblotting and immunohistochemistry were performed using goat polyclonal GAPDH (sc-48166; Santa Cruz Biotechnology, Inc., Dallas, TX), mouse monoclonal TSP1 (sc-393504; Santa Cruz Biotechnology, Inc., Dallas, TX), goat polyclonal TSP2 (sc-12313; Santa Cruz Biotechnology, Inc., Dallas, TX), goat polyclonal CD36 (sc-5522; Santa Cruz Biotechnology, Inc., Dallas, TX), mouse anti-goat IgG-HRP (sc-2354; Santa Cruz Biotechnology, Inc., Dallas, TX), goat anti-mouse IgG-HRP (sc-2005; Santa Cruz Biotechnology, Inc., Dallas, TX) bovine anti-goat IgG-CFL-647 (sc-362284, Lot#B0312) and goat anti-mouse IgG-CFL-647 (sc362257; Santa Cruz Biotechnology, Inc.).

Techniques: Immunohistochemical staining, Microscopy, Imaging, Negative Control

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