Review

sr b1 rabbit polyclonal novus biologicals (Novus Biologicals)

Name: sr b1 rabbit polyclonal novus biologicals
Brand: Novus Biologicals
Rating: 92 (19 reviews)

Novus Biologicals is a verified supplier

Novus Biologicals manufactures this product

About

News

Press Release

Team

Advisors

Partners

Contact

Bioz Stars

Bioz vStars

Buy from Supplier

Structured Review

Novus Biologicals sr b1 rabbit polyclonal novus biologicals
Sr B1 Rabbit Polyclonal Novus Biologicals, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 92/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/sr b1 rabbit polyclonal novus biologicals/product/Novus Biologicals
Average 92 stars, based on 19 article reviews

sr b1 rabbit polyclonal novus biologicals - by Bioz Stars, 2026-03

92/100 stars

Images

Image Search Results

SR-B1 interacts with S1PR1-eGFP in murine macrophages in an HDL-dependent manner. Thioglycolate-elicited peritoneal macrophages were prepared from (A) Sr-b1 WT/WT (C57BL/6J) and Sr-b1 KO/KO mice and (B–D) S1pr1 WT/WT (C57BL/6J) and S1pr1 eGFP/eGFP mice and cultured. A, B: Cells were lysed and protein extracts were prepared and analyzed by immunoblotting for SR-B1, the GFP tag on S1PR1-GFP, and β-actin. C: Macrophages were either untreated or treated in culture with 100 μg (protein)/ml HDL as indicated. Control cells were treated with an equivalent volume of vehicle. After 30 min, cells were fixed, and Duolink proximity ligation assay (PLA) was performed (red fluorescence) as described in the ‘ ’ section. Nuclei were stained using DAPI (blue). C: Representative images (scale bars represent 50 μm; bottom row shows zoomed-in view of the boxed areas of the DAPI + PLA merged images) and (D) quantification of PLA signal performed by counting the proportion of cells exhibiting PLA signal across three fields of view for each sample well. Data are means ± SEM of n = 3 samples. Each data point represents cells isolated from a different mouse. Data were analyzed using the Kruskal-Wallis test and the P -value is indicated. eGFP, enhanced green fluorescent protein; PLA, proximity ligation assay; S1PR, sphingosine-1-phosphate receptor; SR-B1, scavenger receptor class B, type I.

Journal: Journal of Lipid Research

Article Title: Ligand-dependent interactions between SR-B1 and S1PR1 in macrophages and atherosclerotic plaques

doi: 10.1016/j.jlr.2024.100541

Figure Lengend Snippet: SR-B1 interacts with S1PR1-eGFP in murine macrophages in an HDL-dependent manner. Thioglycolate-elicited peritoneal macrophages were prepared from (A) Sr-b1 WT/WT (C57BL/6J) and Sr-b1 KO/KO mice and (B–D) S1pr1 WT/WT (C57BL/6J) and S1pr1 eGFP/eGFP mice and cultured. A, B: Cells were lysed and protein extracts were prepared and analyzed by immunoblotting for SR-B1, the GFP tag on S1PR1-GFP, and β-actin. C: Macrophages were either untreated or treated in culture with 100 μg (protein)/ml HDL as indicated. Control cells were treated with an equivalent volume of vehicle. After 30 min, cells were fixed, and Duolink proximity ligation assay (PLA) was performed (red fluorescence) as described in the ‘ ’ section. Nuclei were stained using DAPI (blue). C: Representative images (scale bars represent 50 μm; bottom row shows zoomed-in view of the boxed areas of the DAPI + PLA merged images) and (D) quantification of PLA signal performed by counting the proportion of cells exhibiting PLA signal across three fields of view for each sample well. Data are means ± SEM of n = 3 samples. Each data point represents cells isolated from a different mouse. Data were analyzed using the Kruskal-Wallis test and the P -value is indicated. eGFP, enhanced green fluorescent protein; PLA, proximity ligation assay; S1PR, sphingosine-1-phosphate receptor; SR-B1, scavenger receptor class B, type I.

Article Snippet: Afterward, the slides were incubated overnight at 4°C with the following primary antibodies: a goat polyclonal anti-GFP antibody (1:100; Abcam Inc Boston MA; catalog # ab6673) and a rabbit polyclonal anti-SR-B1 antibody (1:100; Bio-Techne Canada, Toronto ON, Canada; catalog # NB400-104), and for some experiments, when cells or histological sections were co-stained for either caveolin-1 or Mac3, respectively, with a mouse monoclonal anti-caveolin 1 (1:500; Bio-Techne Canada, Toronto ON, Canada; catalog # NBP3-23193) or a mouse monoclonal anti-Mac3 antibody (1:200; BD Biosciences; catalog # 553322).

Techniques: Cell Culture, Western Blot, Proximity Ligation Assay, Fluorescence, Staining, Isolation

The HDL-stimulated interaction between SR-B1 and S1PR1 is inhibited by S1PL treatment and by inhibition of SR-B1. Thioglycolate-elicited peritoneal macrophages were prepared from S1pr1 eGFP/eGFP mice. A: Cells were incubated for 30 min with HDL that had been pre-treated for 60 min at 37°C with S1PL or with control HDL pre-treated in parallel without S1PL. B: Cells were incubated for 30 min with either S1P (10 nM) or HDL (100 μg protein/ml). C: Cells were pre-treated in culture for 45 min with either an SR-B1 blocking rabbit antiserum or a control, non-immune rabbit antiserum (each at 1.5 μg/ml), as indicated, before the addition of HDL (100 μg protein/ml). D: Cells were pre-treated for 45 min with either BLT-1 (150 nM added in DMSO) or an equivalent amount of DMSO vehicle control. HDL was then added. For each experiment, control cells were also incubated in media in the absence of added HDL (Untreated). After addition of HDL or vehicle, cells were incubated at 37°C for 30 min prior to being washed, fixed, and subjected to the Duolink PLA assay and staining for DAPI. Quantification of PLA signal was performed by counting the proportion of cells exhibiting PLA signal across five fields of view for each sample well. Data are means ± SEM of n = 4 (A) or n = 3 samples (B–D), where each replicate represents cells isolated from a different mouse. Data were analyzed using the Kruskal-Wallis test. P -values are indicated. eGFP, enhanced green fluorescent protein; PLA, proximity ligation assay; S1P, sphingosine-1-phosphate; S1PR, sphingosine-1-phosphate receptor; S1PL, S1PL-lyase; SR-B1, scavenger receptor class B, type I.

Journal: Journal of Lipid Research

Article Title: Ligand-dependent interactions between SR-B1 and S1PR1 in macrophages and atherosclerotic plaques

doi: 10.1016/j.jlr.2024.100541

Figure Lengend Snippet: The HDL-stimulated interaction between SR-B1 and S1PR1 is inhibited by S1PL treatment and by inhibition of SR-B1. Thioglycolate-elicited peritoneal macrophages were prepared from S1pr1 eGFP/eGFP mice. A: Cells were incubated for 30 min with HDL that had been pre-treated for 60 min at 37°C with S1PL or with control HDL pre-treated in parallel without S1PL. B: Cells were incubated for 30 min with either S1P (10 nM) or HDL (100 μg protein/ml). C: Cells were pre-treated in culture for 45 min with either an SR-B1 blocking rabbit antiserum or a control, non-immune rabbit antiserum (each at 1.5 μg/ml), as indicated, before the addition of HDL (100 μg protein/ml). D: Cells were pre-treated for 45 min with either BLT-1 (150 nM added in DMSO) or an equivalent amount of DMSO vehicle control. HDL was then added. For each experiment, control cells were also incubated in media in the absence of added HDL (Untreated). After addition of HDL or vehicle, cells were incubated at 37°C for 30 min prior to being washed, fixed, and subjected to the Duolink PLA assay and staining for DAPI. Quantification of PLA signal was performed by counting the proportion of cells exhibiting PLA signal across five fields of view for each sample well. Data are means ± SEM of n = 4 (A) or n = 3 samples (B–D), where each replicate represents cells isolated from a different mouse. Data were analyzed using the Kruskal-Wallis test. P -values are indicated. eGFP, enhanced green fluorescent protein; PLA, proximity ligation assay; S1P, sphingosine-1-phosphate; S1PR, sphingosine-1-phosphate receptor; S1PL, S1PL-lyase; SR-B1, scavenger receptor class B, type I.

Techniques: Inhibition, Incubation, Blocking Assay, Staining, Isolation, Proximity Ligation Assay

The S1PR1 selective agonist, SEW2871, stimulates and the selective antagonist Ex26 inhibits the interaction between SR-B1 and S1PR1. Thioglycolate-elicited peritoneal macrophages from S1pr1 eGFP/eGFP mice were (A) pre-treated with either the anti-SR-B1 antiserum (1.5 μg/ml) or the SR-B1 inhibitor BLT-1 (150 nM) for 45 min or with control non-immune rabbit serum and DMSO vehicle (where not indicated). After the pre-treatment period, HDL (100 μg protein/ml) or the S1PR1 selective agonist, SEW2871 (1 μM) or no stimulus (untreated) was added for an additional 30 min before cells were fixed. B: Cells were treated with either Ex26 (10 μM, added as a 1,000× stock in DMSO) or DMSO vehicle control for 60 min, at which point HDL (100 μg protein/ml) or vehicle was added and cells were incubated for a further 30 min. After the 30 min incubation with HDL, cells were washed, fixed, and subjected to Duolink PLA and DAPI staining, imaging, and analysis as in the legend to <xref ref-type=

Figs. 1 and . Each symbol represents cells isolated from a different mouse. Data were analyzed using Kruskal-Wallis test; P -values are indicated above each graph. C: Thioglycolate-elicited peritoneal macrophages from S1pr1 eGFP/eGFP mice were incubated for 60 min with 10 μM Ex26 or DMSO vehicle control followed by treatment without or with HDL (100 μg protein/ml) for 30 min as indicated. Cells were then washed and fixed, and PLA staining (red) was carried out followed by staining for caveolin-1 using a mouse anti-caveolin-1 antibody and Alexa488-conjugated anti-mouse secondary antibody (green) and DAPI staining (blue). Panels on the right show zoomed-in views of the boxed areas of the merged images of HDL-treated control and Ex26-treated cells. eGFP, enhanced green fluorescent protein; PLA, proximity ligation assay; S1PR, sphingosine-1-phosphate receptor; SR-B1, scavenger receptor class B, type I. " width="100%" height="100%">

Journal: Journal of Lipid Research

Article Title: Ligand-dependent interactions between SR-B1 and S1PR1 in macrophages and atherosclerotic plaques

doi: 10.1016/j.jlr.2024.100541

Figure Lengend Snippet: The S1PR1 selective agonist, SEW2871, stimulates and the selective antagonist Ex26 inhibits the interaction between SR-B1 and S1PR1. Thioglycolate-elicited peritoneal macrophages from S1pr1 eGFP/eGFP mice were (A) pre-treated with either the anti-SR-B1 antiserum (1.5 μg/ml) or the SR-B1 inhibitor BLT-1 (150 nM) for 45 min or with control non-immune rabbit serum and DMSO vehicle (where not indicated). After the pre-treatment period, HDL (100 μg protein/ml) or the S1PR1 selective agonist, SEW2871 (1 μM) or no stimulus (untreated) was added for an additional 30 min before cells were fixed. B: Cells were treated with either Ex26 (10 μM, added as a 1,000× stock in DMSO) or DMSO vehicle control for 60 min, at which point HDL (100 μg protein/ml) or vehicle was added and cells were incubated for a further 30 min. After the 30 min incubation with HDL, cells were washed, fixed, and subjected to Duolink PLA and DAPI staining, imaging, and analysis as in the legend to Figs. 1 and . Each symbol represents cells isolated from a different mouse. Data were analyzed using Kruskal-Wallis test; P -values are indicated above each graph. C: Thioglycolate-elicited peritoneal macrophages from S1pr1 eGFP/eGFP mice were incubated for 60 min with 10 μM Ex26 or DMSO vehicle control followed by treatment without or with HDL (100 μg protein/ml) for 30 min as indicated. Cells were then washed and fixed, and PLA staining (red) was carried out followed by staining for caveolin-1 using a mouse anti-caveolin-1 antibody and Alexa488-conjugated anti-mouse secondary antibody (green) and DAPI staining (blue). Panels on the right show zoomed-in views of the boxed areas of the merged images of HDL-treated control and Ex26-treated cells. eGFP, enhanced green fluorescent protein; PLA, proximity ligation assay; S1PR, sphingosine-1-phosphate receptor; SR-B1, scavenger receptor class B, type I.

Techniques: Incubation, Staining, Imaging, Isolation, Proximity Ligation Assay

The SR-B1 inhibitor, BLT-1, the S1PR1 antagonist Ex26, and S1PL treatment of HDL all reduce HDL-mediated cholesterol efflux from macrophages. Thioglycolate-elicited peritoneal macrophages were collected from wild type C57BL/6J mice, cultured in DMEM containing 10% FBS, 2 mM L-glutamine, 50 μg/ml penicillin, and 50 U/ml streptomycin, for 16 h prior to changing the medium to phenol red-free DMEM containing 3% newborn calf lipoprotein-deficient serum, 2 mM L-glutamine, 50 μg/ml penicillin, and 50 U/ml streptomycin, for an additional 16 h as described above. Cholesterol efflux was measured using the cell-based fluorescent Cholesterol Efflux Assay kit. After loading cells with the fluorescent tracer for 1 h, cells were washed and efflux was initiated by addition of the efflux acceptor medium. Efflux was monitored using either no acceptor or control- or S1PL-treated HDL (each at 100 μg protein/ml) as cholesterol acceptors. Some cells were also treated with Ex26 (10 μM) or BLT-1 (150 nM) during efflux. Cholesterol efflux was measured as the amount of cholesterol tracer appearing in the medium at the end of 3 h, as a % of the total cholesterol tracer (cells + medium). Each symbol represents cells isolated from a different mouse (n = 4); bars represent means and error bars represent standard errors. Data was analyzed by the Kruskal-Wallis test; P value is indicated. S1PL, S1PL-lyase; S1PR, sphingosine-1-phosphate receptor; SR-B1, scavenger receptor class B, type I.

Journal: Journal of Lipid Research

Article Title: Ligand-dependent interactions between SR-B1 and S1PR1 in macrophages and atherosclerotic plaques

doi: 10.1016/j.jlr.2024.100541

Figure Lengend Snippet: The SR-B1 inhibitor, BLT-1, the S1PR1 antagonist Ex26, and S1PL treatment of HDL all reduce HDL-mediated cholesterol efflux from macrophages. Thioglycolate-elicited peritoneal macrophages were collected from wild type C57BL/6J mice, cultured in DMEM containing 10% FBS, 2 mM L-glutamine, 50 μg/ml penicillin, and 50 U/ml streptomycin, for 16 h prior to changing the medium to phenol red-free DMEM containing 3% newborn calf lipoprotein-deficient serum, 2 mM L-glutamine, 50 μg/ml penicillin, and 50 U/ml streptomycin, for an additional 16 h as described above. Cholesterol efflux was measured using the cell-based fluorescent Cholesterol Efflux Assay kit. After loading cells with the fluorescent tracer for 1 h, cells were washed and efflux was initiated by addition of the efflux acceptor medium. Efflux was monitored using either no acceptor or control- or S1PL-treated HDL (each at 100 μg protein/ml) as cholesterol acceptors. Some cells were also treated with Ex26 (10 μM) or BLT-1 (150 nM) during efflux. Cholesterol efflux was measured as the amount of cholesterol tracer appearing in the medium at the end of 3 h, as a % of the total cholesterol tracer (cells + medium). Each symbol represents cells isolated from a different mouse (n = 4); bars represent means and error bars represent standard errors. Data was analyzed by the Kruskal-Wallis test; P value is indicated. S1PL, S1PL-lyase; S1PR, sphingosine-1-phosphate receptor; SR-B1, scavenger receptor class B, type I.

Techniques: Cell Culture, Isolation

HDL-mediated protection of macrophages from tunicamycin-induced apoptosis is inhibited by the SR-B1 inhibitor BLT-1 and by S1PL-treatment of HDL. Thioglycolate-elicited peritoneal macrophages from wild type C57BL/6J mice were cultured in 8-well chamber slides in DMEM containing 3% newborn calf lipoprotein-deficient serum for 16 h prior to the addition of (A, B) tunicamycin (10 μg/ml) without or with HDL (50 μg protein/ml), (C) tunicamycin (10 μg/ml), HDL (50 μg protein/ml), and BLT-1 (150 nM) or combinations of those as indicated; or (D) tunicamycin (10 μg/ml) and either S1PL-treated or control-treated HDL (50 μg protein/ml) as indicated. After 24 h, cells were fixed and apoptosis was detected by TUNEL staining for fragmented DNA, followed by counter-staining with DAPI for nuclear DNA. A: Representative images of TUNEL (green fluorescence) and DAPI (blue fluorescence) stained nuclei. B–D: Quantification of the degree of apoptosis (% TUNEL-positive nuclei). Each data point in (B) represents cells isolated from a different mouse (n = 4). Data points in (C) and (D) represent independent wells of cells isolated from 2 to 4 mice, plated in triplicate. Data were analyzed by the Kruskal-Wallis test; P values are indicated above each graph. S1PL, S1PL-lyase; TUNEL, Terminal deoxynucleotidyl transferase-mediated dUTP Nick End Labeling; SR-B1, scavenger receptor class B, type I.

Journal: Journal of Lipid Research

Article Title: Ligand-dependent interactions between SR-B1 and S1PR1 in macrophages and atherosclerotic plaques

doi: 10.1016/j.jlr.2024.100541

Figure Lengend Snippet: HDL-mediated protection of macrophages from tunicamycin-induced apoptosis is inhibited by the SR-B1 inhibitor BLT-1 and by S1PL-treatment of HDL. Thioglycolate-elicited peritoneal macrophages from wild type C57BL/6J mice were cultured in 8-well chamber slides in DMEM containing 3% newborn calf lipoprotein-deficient serum for 16 h prior to the addition of (A, B) tunicamycin (10 μg/ml) without or with HDL (50 μg protein/ml), (C) tunicamycin (10 μg/ml), HDL (50 μg protein/ml), and BLT-1 (150 nM) or combinations of those as indicated; or (D) tunicamycin (10 μg/ml) and either S1PL-treated or control-treated HDL (50 μg protein/ml) as indicated. After 24 h, cells were fixed and apoptosis was detected by TUNEL staining for fragmented DNA, followed by counter-staining with DAPI for nuclear DNA. A: Representative images of TUNEL (green fluorescence) and DAPI (blue fluorescence) stained nuclei. B–D: Quantification of the degree of apoptosis (% TUNEL-positive nuclei). Each data point in (B) represents cells isolated from a different mouse (n = 4). Data points in (C) and (D) represent independent wells of cells isolated from 2 to 4 mice, plated in triplicate. Data were analyzed by the Kruskal-Wallis test; P values are indicated above each graph. S1PL, S1PL-lyase; TUNEL, Terminal deoxynucleotidyl transferase-mediated dUTP Nick End Labeling; SR-B1, scavenger receptor class B, type I.

Techniques: Cell Culture, TUNEL Assay, Staining, Fluorescence, Isolation, End Labeling

SR-B1 and S1PR1 interact in atherosclerotic plaques of high-fat diet-fed ApoE KO/KO mice. Control ApoE KO/KO mice in which S1PR1 was not GFP tagged ( S1pr1 WT/WT / ApoE KO/KO mice), and S1pr1 eGFP/eGFP ApoE KO/KO mice were fed a high fat, high cholesterol diet for 8 weeks, beginning at 15 weeks of age. S1pr1 eGFP/eGFP / ApoE KO/KO mice were then treated with Ex26 (30 mg/kg in DMSO) or DMSO vehicle and mice were euthanized, and tissues were harvested 12 h later. Adjacent cross-sections in the aortic sinus were collected and stained with oil red O (lipid) and hematoxylin (nuclei) to detect lipid-rich atherosclerotic plaques or were fixed, permeabilized, and subjected to Duolink PLA staining using antibodies against SR-B1 and GFP as described in the ‘ ’ section. PLA-stained sections were counterstained with DAPI for nuclear DNA. A: Representative images of oil red O and hematoxylin-stained atherosclerotic plaques in the aortic sinuses. B: Images of adjacent PLA-stained sections of plaques showing DAPI, PLA signal, and merged DAPI and PLA signals. Scale bars (A, B) represent 20 μm. C: Quantification of PLA signal in the atherosclerotic plaques of S1pr1 WT/WT /ApoE KO/KO and DMSO vehicle or Ex26-treated S1pr1 eGFP/eGFP /ApoE KO/KO mice was performed by counting the average of PLA signal punctae within atherosclerotic plaques across three fields of view per plaque of three sections for each mouse and dividing by the number of DAPI-stained nuclei. Each data point represents data from a different mouse. Data were analyzed using the Kruskal-Wallis test; P value is indicated above the graph. D: Confocal images of PLA (red) and anti-Mac3 immunofluorescence (green) and DAPI (blue) co-stained images of atherosclerotic plaques from control S1pr1 WT/WT /ApoE KO/KO and DMSO vehicle or Ex26-treated S1pr1 eGFP/eGFP /ApoE KO/KO mice. Images in the bottom row correspond to zoomed-in views of the boxed areas. eGFP, enhanced green fluorescent protein; PLA, proximity ligation assay; S1PR, sphingosine-1-phosphate receptor; SR-B1, scavenger receptor class B, type I.

Journal: Journal of Lipid Research

Article Title: Ligand-dependent interactions between SR-B1 and S1PR1 in macrophages and atherosclerotic plaques

doi: 10.1016/j.jlr.2024.100541

Figure Lengend Snippet: SR-B1 and S1PR1 interact in atherosclerotic plaques of high-fat diet-fed ApoE KO/KO mice. Control ApoE KO/KO mice in which S1PR1 was not GFP tagged ( S1pr1 WT/WT / ApoE KO/KO mice), and S1pr1 eGFP/eGFP ApoE KO/KO mice were fed a high fat, high cholesterol diet for 8 weeks, beginning at 15 weeks of age. S1pr1 eGFP/eGFP / ApoE KO/KO mice were then treated with Ex26 (30 mg/kg in DMSO) or DMSO vehicle and mice were euthanized, and tissues were harvested 12 h later. Adjacent cross-sections in the aortic sinus were collected and stained with oil red O (lipid) and hematoxylin (nuclei) to detect lipid-rich atherosclerotic plaques or were fixed, permeabilized, and subjected to Duolink PLA staining using antibodies against SR-B1 and GFP as described in the ‘ ’ section. PLA-stained sections were counterstained with DAPI for nuclear DNA. A: Representative images of oil red O and hematoxylin-stained atherosclerotic plaques in the aortic sinuses. B: Images of adjacent PLA-stained sections of plaques showing DAPI, PLA signal, and merged DAPI and PLA signals. Scale bars (A, B) represent 20 μm. C: Quantification of PLA signal in the atherosclerotic plaques of S1pr1 WT/WT /ApoE KO/KO and DMSO vehicle or Ex26-treated S1pr1 eGFP/eGFP /ApoE KO/KO mice was performed by counting the average of PLA signal punctae within atherosclerotic plaques across three fields of view per plaque of three sections for each mouse and dividing by the number of DAPI-stained nuclei. Each data point represents data from a different mouse. Data were analyzed using the Kruskal-Wallis test; P value is indicated above the graph. D: Confocal images of PLA (red) and anti-Mac3 immunofluorescence (green) and DAPI (blue) co-stained images of atherosclerotic plaques from control S1pr1 WT/WT /ApoE KO/KO and DMSO vehicle or Ex26-treated S1pr1 eGFP/eGFP /ApoE KO/KO mice. Images in the bottom row correspond to zoomed-in views of the boxed areas. eGFP, enhanced green fluorescent protein; PLA, proximity ligation assay; S1PR, sphingosine-1-phosphate receptor; SR-B1, scavenger receptor class B, type I.

Techniques: Staining, Immunofluorescence, Proximity Ligation Assay

(A) Caco-2/TC7 cell homogenates (150 µg) were subjected to SDS-PAGE and transferred onto nitrocellulose membranes. Membranes were incubated with PPM or IPM containing biotinylated phospholipids coupled to horseradish-streptavidin peroxidase and developed using the ECL system. (B) Caco-2/TC7 cell homogenates were immunoprecipitated with anti-SR-BI antibodies or non-immune serum and the resulting protein samples were subjected to SDS-PAGE and transferred onto nitrocellulose membrane. The membrane was incubated with PPM as in (A). NI, non immune serum; NS, non specific signal.

Journal: PLoS ONE

Article Title: Sensing of Dietary Lipids by Enterocytes: A New Role for SR-BI/CLA-1

doi: 10.1371/journal.pone.0004278

Figure Lengend Snippet: (A) Caco-2/TC7 cell homogenates (150 µg) were subjected to SDS-PAGE and transferred onto nitrocellulose membranes. Membranes were incubated with PPM or IPM containing biotinylated phospholipids coupled to horseradish-streptavidin peroxidase and developed using the ECL system. (B) Caco-2/TC7 cell homogenates were immunoprecipitated with anti-SR-BI antibodies or non-immune serum and the resulting protein samples were subjected to SDS-PAGE and transferred onto nitrocellulose membrane. The membrane was incubated with PPM as in (A). NI, non immune serum; NS, non specific signal.

Article Snippet: Antibodies used were: mouse polyclonal anti-Cla-1 (BD Transduction Laboratories), rabbit polyclonal anti-SR-BI (Novus 400-104), sheep polyclonal anti-PLAP (ABDSerotec 0300-1909), rabbit polyclonal anti-ERK (Cell Signaling), rabbit polyclonal anti-P-ERK (Thr202/Tyr204, Cell Signaling), rabbit polyclonal anti-p38 MAPK (Cell Signaling), rabbit polyclonal anti-P-p38 MAPK (Thr180/Tyr182, Cell Signaling), mouse monoclonal anti-E-cadh (BD transduction laboratories), goat polyclonal anti-EEA1 (Santa Cruz Biotechnology), mouse monoclonal anti-flotillin-1 (BD Transduction Laboratories), mouse monoclonal anti-sucrase-isomaltase (provided by Dr.

Techniques: SDS Page, Incubation, Immunoprecipitation

$(A) Immunoelectron micrograph of SR-BI/CLA-1 in untreated differentiated Caco-2/TC7 cells. MV, microvilli; TW, terminal web (bar, 0.5 µm). Note the significant amount of intracellular trafficking SR-BI/CLA-1 in addition to its main apical localization (arrowheads). (B) Immunolocalization of SR-BI/CLA-1 (green channel) and sucrase isomaltase (SI, red channel) in differentiated Caco-2/TC7 cells before (T0) and after 5, 10 and 15 min of apical PPM supply. Panels represent XY acquisitions at the apical level (bar, 10 µm). Arrowheads show clusters of SR-BI/CLA-1. (C) Immunolocalization of SR-BI/CLA-1 in differentiated Caco-2/TC7 cells in the absence (control) or presence of PPM or IPM for 20 min (bar, 20 µm). Arrowheads show clusters of SR-BI/CLA-1 (D) Immunoelectron micrograph of SR-BI/CLA-1 in Caco-2/TC7 cells supplied with PPM (MV, microvilli). Arrowheads indicate SR-BI/CLA-1 clusters (bar, 100 nm). (E) Cell surface biotinylation assay for apical SR-BI/CLA-1. Caco-2/TC7 cells were cultured in the absence (0) or presence of PPM for the indicated times. Cells were then selectively labeled with non-permeant biotin at the apical (left panel) or basal surface (right panel). Biotinylated fractions were obtained as described in . Total cell lysates (total), apical and basal biotinylated fractions (left and right panel respectively) and non-apical fractions (non-apical) were analyzed in immunoblots of SR-BI/CLA-1, E-cadherin being used as a basolateral membrane marker.$

Journal: PLoS ONE

Article Title: Sensing of Dietary Lipids by Enterocytes: A New Role for SR-BI/CLA-1

doi: 10.1371/journal.pone.0004278

Figure Lengend Snippet: (A) Immunoelectron micrograph of SR-BI/CLA-1 in untreated differentiated Caco-2/TC7 cells. MV, microvilli; TW, terminal web (bar, 0.5 µm). Note the significant amount of intracellular trafficking SR-BI/CLA-1 in addition to its main apical localization (arrowheads). (B) Immunolocalization of SR-BI/CLA-1 (green channel) and sucrase isomaltase (SI, red channel) in differentiated Caco-2/TC7 cells before (T0) and after 5, 10 and 15 min of apical PPM supply. Panels represent XY acquisitions at the apical level (bar, 10 µm). Arrowheads show clusters of SR-BI/CLA-1. (C) Immunolocalization of SR-BI/CLA-1 in differentiated Caco-2/TC7 cells in the absence (control) or presence of PPM or IPM for 20 min (bar, 20 µm). Arrowheads show clusters of SR-BI/CLA-1 (D) Immunoelectron micrograph of SR-BI/CLA-1 in Caco-2/TC7 cells supplied with PPM (MV, microvilli). Arrowheads indicate SR-BI/CLA-1 clusters (bar, 100 nm). (E) Cell surface biotinylation assay for apical SR-BI/CLA-1. Caco-2/TC7 cells were cultured in the absence (0) or presence of PPM for the indicated times. Cells were then selectively labeled with non-permeant biotin at the apical (left panel) or basal surface (right panel). Biotinylated fractions were obtained as described in . Total cell lysates (total), apical and basal biotinylated fractions (left and right panel respectively) and non-apical fractions (non-apical) were analyzed in immunoblots of SR-BI/CLA-1, E-cadherin being used as a basolateral membrane marker.

Techniques: Cell Surface Biotinylation Assay, Cell Culture, Labeling, Western Blot, Marker

$(A) Caco-2/TC7 cells were harvested in the presence of Triton X-100 and the lysate fractionated on a 5–40% sucrose gradient. Eleven fractions were collected for immunoblots of SR-BI/CLA-1, EEA1 (early endosome antigen 1) and flottilin-1 (raft marker). (B) Caco-2/TC7 cells were cultured in the absence (control) or presence of PPM or IPM for 10 min and then harvested in the presence of Triton X-100. Cell lysates were applied to a 5–40% sucrose gradient and eleven fractions collected. Fractions 3 to 8 were analyzed by immunoblotting with antibodies against SR-BI/CLA-1 (left panel) and flottilin-1 (right panel). (C) Immunolocalization of SR-BI/CLA-1 and alkaline phosphatase (PLAP, used as raft marker) in the brush border of Caco-2/TC7 cells supplied with PPM. SR-BI/CLA-1 is labelled with anti-rabbit immunoglobulin-gold complexes (18-nm particles) and PLAP with anti-sheep immunoglobulin-gold complexes (12-nm particles). MV, microvilli; bar, 100 nm.$

Journal: PLoS ONE

Article Title: Sensing of Dietary Lipids by Enterocytes: A New Role for SR-BI/CLA-1

doi: 10.1371/journal.pone.0004278

Figure Lengend Snippet: (A) Caco-2/TC7 cells were harvested in the presence of Triton X-100 and the lysate fractionated on a 5–40% sucrose gradient. Eleven fractions were collected for immunoblots of SR-BI/CLA-1, EEA1 (early endosome antigen 1) and flottilin-1 (raft marker). (B) Caco-2/TC7 cells were cultured in the absence (control) or presence of PPM or IPM for 10 min and then harvested in the presence of Triton X-100. Cell lysates were applied to a 5–40% sucrose gradient and eleven fractions collected. Fractions 3 to 8 were analyzed by immunoblotting with antibodies against SR-BI/CLA-1 (left panel) and flottilin-1 (right panel). (C) Immunolocalization of SR-BI/CLA-1 and alkaline phosphatase (PLAP, used as raft marker) in the brush border of Caco-2/TC7 cells supplied with PPM. SR-BI/CLA-1 is labelled with anti-rabbit immunoglobulin-gold complexes (18-nm particles) and PLAP with anti-sheep immunoglobulin-gold complexes (12-nm particles). MV, microvilli; bar, 100 nm.

Techniques: Western Blot, Marker, Cell Culture

(A) Caco-2/TC7 Cell populations 63 and 64, expressing lentiviral shRNA 63 and 64 respectively, were analyzed at passage 4 after transfection in the absence of PPM or after 10 min of PPM supply. Cell lysates were analyzed by immunoblot with antibodies against SR-BI/CLA-1 and E-cadherin (E-cadh, used as loading control). The lower panel shows the level of SR-BI/CLA-1 expression normalized to the level of E-cadherin expression set at 100% for control Caco-2/TC7 cells. Results are from two independent sets of experiments. (B) Cell populations 63 and 64 were cultured on semi-permeable filters and incubated in the absence or presence of PPM or IPM in the apical compartment for the indicated times. An early (63E) and a late (63L) passage (corresponding respectively to passage 6 and 28 after transfection) of Cell population 63 were compared to Cell population 64 at passage 28. Cell lysates were analyzed in SR-BI/CLA-1 and phospho-ERK1/2 (P-ERK) immunoblots. Total ERK (ERK) and E-cadherin (E-cadh) were used as loading controls. Lower panel, the ratio of P-ERK expression normalized to total ERK expression in PPM-treated cells versus IPM-treated cells, set at 100% for Cell population 64. Results show the means±SEM of three independent sets of experiments. *P<0.05 compared to Cell population 64. (C) Control Caco-2/TC7 cells and an early passage of Cell population 63 (passage 7) were pre-incubated for 1 hour with cycloheximide before PPM were added for 20 min more. Immunolocalization of apoB in these cells was performed using concanavalin A (conA) to visualize the apical membrane. Upper panels show the merged labeling of conA (green) and apoB (blue) in the apical plane (XY planes; bar, 50 µm), lower panels show the same apical XY planes with apoB alone (white). Note the absence of apoB from the apical compartment of control cells after PPM supply resulting from its chase towards intracellular domains and the persistence of apoB at the apical pole of SR-BI/CLA-1-knockdown cells.

Journal: PLoS ONE

Article Title: Sensing of Dietary Lipids by Enterocytes: A New Role for SR-BI/CLA-1

doi: 10.1371/journal.pone.0004278

Figure Lengend Snippet: (A) Caco-2/TC7 Cell populations 63 and 64, expressing lentiviral shRNA 63 and 64 respectively, were analyzed at passage 4 after transfection in the absence of PPM or after 10 min of PPM supply. Cell lysates were analyzed by immunoblot with antibodies against SR-BI/CLA-1 and E-cadherin (E-cadh, used as loading control). The lower panel shows the level of SR-BI/CLA-1 expression normalized to the level of E-cadherin expression set at 100% for control Caco-2/TC7 cells. Results are from two independent sets of experiments. (B) Cell populations 63 and 64 were cultured on semi-permeable filters and incubated in the absence or presence of PPM or IPM in the apical compartment for the indicated times. An early (63E) and a late (63L) passage (corresponding respectively to passage 6 and 28 after transfection) of Cell population 63 were compared to Cell population 64 at passage 28. Cell lysates were analyzed in SR-BI/CLA-1 and phospho-ERK1/2 (P-ERK) immunoblots. Total ERK (ERK) and E-cadherin (E-cadh) were used as loading controls. Lower panel, the ratio of P-ERK expression normalized to total ERK expression in PPM-treated cells versus IPM-treated cells, set at 100% for Cell population 64. Results show the means±SEM of three independent sets of experiments. *P<0.05 compared to Cell population 64. (C) Control Caco-2/TC7 cells and an early passage of Cell population 63 (passage 7) were pre-incubated for 1 hour with cycloheximide before PPM were added for 20 min more. Immunolocalization of apoB in these cells was performed using concanavalin A (conA) to visualize the apical membrane. Upper panels show the merged labeling of conA (green) and apoB (blue) in the apical plane (XY planes; bar, 50 µm), lower panels show the same apical XY planes with apoB alone (white). Note the absence of apoB from the apical compartment of control cells after PPM supply resulting from its chase towards intracellular domains and the persistence of apoB at the apical pole of SR-BI/CLA-1-knockdown cells.

Techniques: Expressing, shRNA, Transfection, Western Blot, Cell Culture, Incubation, Labeling

P. berghei sporozoites can infect primary mouse hepatocytes through CD81-and SR-B1-independent pathways. ( A ) Primary hepatocytes isolated from WT or SR-B1 deficient C57BL/6 mice were infected with PbGFP sporozoites in the absence or presence of neutralizing anti-mCD81 mAb MT81, and cultured for 24 h before EEF quantification (mean control values for each experiment without MT81: 63 and 266 EEFs/well in srb1 +/+ hepatocytes; 94, 311 and 420 EEFs/well in srb1 −/− hepatocytes). * p < 0.05 (ratio paired t test). (B) P. berghei infection in Hepa1-6 cells was inhibited by the anti-mCD81 mAb MT81 antibody (mean control values for each experiment without MT81: 0.64 and 1.09% PbGFP-infected cells).

Journal: Scientific Reports

Article Title: Molecular determinants of SR-B1-dependent Plasmodium sporozoite entry into hepatocytes

doi: 10.1038/s41598-020-70468-2

Figure Lengend Snippet: P. berghei sporozoites can infect primary mouse hepatocytes through CD81-and SR-B1-independent pathways. ( A ) Primary hepatocytes isolated from WT or SR-B1 deficient C57BL/6 mice were infected with PbGFP sporozoites in the absence or presence of neutralizing anti-mCD81 mAb MT81, and cultured for 24 h before EEF quantification (mean control values for each experiment without MT81: 63 and 266 EEFs/well in srb1 +/+ hepatocytes; 94, 311 and 420 EEFs/well in srb1 −/− hepatocytes). * p < 0.05 (ratio paired t test). (B) P. berghei infection in Hepa1-6 cells was inhibited by the anti-mCD81 mAb MT81 antibody (mean control values for each experiment without MT81: 0.64 and 1.09% PbGFP-infected cells).

Article Snippet: All incubations were performed at 4 °C in PBS supplemented with 3% (v/v) BSA for one hour with either “αH” anti-SR-B1 polyclonal rabbit serum or “αM” anti-SR-B1 polyclonal rabbit antibodies NB400-113 (Novus Biological).

Techniques: Isolation, Infection, Cell Culture, Control

Mouse SR-B1 poorly supports P. berghei sporozoite invasion. (A,B) CD81KOH16 cells were transfected with either mouse or human SR-B1 plasmids, or no plasmid as a control (Mock). Total protein expression was analyzed using polyclonal anti-SR-B1 antibodies (Ab24603) by western blot (A) with GAPDH as a loading control. Surface protein expression was analyzed by flow cytometry (B) using anti-human “αH” SR-B1 polyclonal rabbit serum (blue) and anti-mouse “αM” polyclonal antibodies NB400-113 (orange). The grey histogram represents untransfected cells labeled with the corresponding antibody. (C,D) CD81KOH16 (C) and WT Hepa1-6 cells treated with siRNA against CD81 24 h before (D) , were transfected with mouse or human SR-B1 plasmids, or no plasmid as a negative control (Mock), and then infected with PbGFP sporozoites. EEF numbers were counted by microscopy after UIS4 staining, 24 h after sporozoite addition. The mean control values for each experiment were 59, 139, 214, 245, 299, 315 and 383 EEFs/well in hSR-B1-transfected CD81KOH16 cells (C) , and 30, 140, 155 and 215 EEFs/well in control Hepa1-6 cells (D) . * p < 0.05; ** p < 0.01 (repeated measures one-way ANOVA followed by Tukey’s multiple comparisons test).

Journal: Scientific Reports

Article Title: Molecular determinants of SR-B1-dependent Plasmodium sporozoite entry into hepatocytes

doi: 10.1038/s41598-020-70468-2

Figure Lengend Snippet: Mouse SR-B1 poorly supports P. berghei sporozoite invasion. (A,B) CD81KOH16 cells were transfected with either mouse or human SR-B1 plasmids, or no plasmid as a control (Mock). Total protein expression was analyzed using polyclonal anti-SR-B1 antibodies (Ab24603) by western blot (A) with GAPDH as a loading control. Surface protein expression was analyzed by flow cytometry (B) using anti-human “αH” SR-B1 polyclonal rabbit serum (blue) and anti-mouse “αM” polyclonal antibodies NB400-113 (orange). The grey histogram represents untransfected cells labeled with the corresponding antibody. (C,D) CD81KOH16 (C) and WT Hepa1-6 cells treated with siRNA against CD81 24 h before (D) , were transfected with mouse or human SR-B1 plasmids, or no plasmid as a negative control (Mock), and then infected with PbGFP sporozoites. EEF numbers were counted by microscopy after UIS4 staining, 24 h after sporozoite addition. The mean control values for each experiment were 59, 139, 214, 245, 299, 315 and 383 EEFs/well in hSR-B1-transfected CD81KOH16 cells (C) , and 30, 140, 155 and 215 EEFs/well in control Hepa1-6 cells (D) . * p < 0.05; ** p < 0.01 (repeated measures one-way ANOVA followed by Tukey’s multiple comparisons test).

Techniques: Transfection, Plasmid Preparation, Control, Expressing, Western Blot, Flow Cytometry, Labeling, Negative Control, Infection, Microscopy, Staining

SR-B1 modeling identifies potential functional regions. (A) Predicted tertiary structure of hSR-B1 extracellular domain by homology modeling using CD36 (PDB ID: 5lgd) as a template, with the three regions referred to as “N-terminal” (green), “apex” (red) and “C-terminal” (black). (B) A close-up view of structural alignment of the apical helix bundle of mouse (orange) and human (blue) SR-B1, with their four alpha helices (α4 to α7). The main structural differences are circled in black. (C) Schematic representation of SR-B1 N-glycosylation sites on human (blue) and mouse (orange) proteins. Two determinant sites for SR-B1 structure and function are in red (Asn 108 and 173), mouse specific sites are in yellow (Asn 116 and 288) and conserved sites are in blue. SR-B1 model is a schematic representation of the delineated regions (“N-terminal” (green), “apex” (red), “C-terminal” (black)) in SR-B1 protein displaying all potential N-glycosylation sites. (D) Pairwise sequence alignment of mSR-B1 and hSR-B1 proteins for the 132–223 apical region with corresponding predicted human secondary structure (alpha helices in red and beta strand in blue). Identical, similar and different amino acids are represented in black, blue and red respectively. The threonine residue position corresponding to PfEMP1 binding phenylalanine in CD36 homolog is highlighted in purple. The residues in SR-B1 equivalent to Enterovirus-interacting site in LIMP-2 are highlighted in green and purple. (E) Electrostatic surface potential of mSR-B1 and hSR-B1 extracellular domain from side and top views. Values are in units of kT/e at 298 K, on a scale of − 2 kT/e (red) to + 2 kT/e (blue). White color indicates a neutral potential. The black circle highlights a differential electrostatic surface potential between mSR-B1 and hSR-B1 at the top of the “apex” region.

Journal: Scientific Reports

Article Title: Molecular determinants of SR-B1-dependent Plasmodium sporozoite entry into hepatocytes

doi: 10.1038/s41598-020-70468-2

Figure Lengend Snippet: SR-B1 modeling identifies potential functional regions. (A) Predicted tertiary structure of hSR-B1 extracellular domain by homology modeling using CD36 (PDB ID: 5lgd) as a template, with the three regions referred to as “N-terminal” (green), “apex” (red) and “C-terminal” (black). (B) A close-up view of structural alignment of the apical helix bundle of mouse (orange) and human (blue) SR-B1, with their four alpha helices (α4 to α7). The main structural differences are circled in black. (C) Schematic representation of SR-B1 N-glycosylation sites on human (blue) and mouse (orange) proteins. Two determinant sites for SR-B1 structure and function are in red (Asn 108 and 173), mouse specific sites are in yellow (Asn 116 and 288) and conserved sites are in blue. SR-B1 model is a schematic representation of the delineated regions (“N-terminal” (green), “apex” (red), “C-terminal” (black)) in SR-B1 protein displaying all potential N-glycosylation sites. (D) Pairwise sequence alignment of mSR-B1 and hSR-B1 proteins for the 132–223 apical region with corresponding predicted human secondary structure (alpha helices in red and beta strand in blue). Identical, similar and different amino acids are represented in black, blue and red respectively. The threonine residue position corresponding to PfEMP1 binding phenylalanine in CD36 homolog is highlighted in purple. The residues in SR-B1 equivalent to Enterovirus-interacting site in LIMP-2 are highlighted in green and purple. (E) Electrostatic surface potential of mSR-B1 and hSR-B1 extracellular domain from side and top views. Values are in units of kT/e at 298 K, on a scale of − 2 kT/e (red) to + 2 kT/e (blue). White color indicates a neutral potential. The black circle highlights a differential electrostatic surface potential between mSR-B1 and hSR-B1 at the top of the “apex” region.

Techniques: Functional Assay, Glycoproteomics, Sequencing, Residue, Binding Assay

The apical domain of SR-B1 plays a crucial role during P. berghei infection. (A) Schematic representation of the ApicalH and ApicalM chimeric constructs. (B) Predicted tertiary structure of ApicalH and ApicalM chimeras by homology modeling, highlighting the portions of mouse (orange) or human (blue) origins. (C) Top views of the electrostatic surface potential of ApicalH and ApicalM chimeras’ apex. Values are in units of kT/e at 298 K, on a scale of − 2 kT/e (red) to + 2 kT/e (blue). White color indicates a neutral potential. The black circle highlights a differential electrostatic surface potential between the two chimeric constructs at the top of the “apex” region. (D) CD81KOH16 cells were transfected with hSR-B1, mSR-B1, ApicalH or ApicalM chimera plasmids, or no plasmid as a control (Mock). Protein surface expression was analyzed using anti-hSR-B1 (“αH”, blue histograms) and anti-mSR-B1 (“αM”, orange histograms), 24 h after transfection. The grey histogram represents untransfected cells stained with the cognate antibody. (E) CD81KOH16 cells were transfected with hSR-B1, mSR-B1, ApicalH or ApicalM constructs, or no plasmid as a control (Mock), and infected with PbGFP sporozoites 24 h after transfection. The number of infected cells (EEFs) was determined by microscopy after UIS4 staining, 24 h after sporozoite addition (mean control values for each experiment: 59, 139, 214, 245, 299, 315 and 383 EEFs/well in hSR-B1-transfected CD81KOH16 cells). ns, non-significant; *** p < 0.001 (one-way ANOVA followed by Tukey’s multiple comparisons test).

Journal: Scientific Reports

Article Title: Molecular determinants of SR-B1-dependent Plasmodium sporozoite entry into hepatocytes

doi: 10.1038/s41598-020-70468-2

Figure Lengend Snippet: The apical domain of SR-B1 plays a crucial role during P. berghei infection. (A) Schematic representation of the ApicalH and ApicalM chimeric constructs. (B) Predicted tertiary structure of ApicalH and ApicalM chimeras by homology modeling, highlighting the portions of mouse (orange) or human (blue) origins. (C) Top views of the electrostatic surface potential of ApicalH and ApicalM chimeras’ apex. Values are in units of kT/e at 298 K, on a scale of − 2 kT/e (red) to + 2 kT/e (blue). White color indicates a neutral potential. The black circle highlights a differential electrostatic surface potential between the two chimeric constructs at the top of the “apex” region. (D) CD81KOH16 cells were transfected with hSR-B1, mSR-B1, ApicalH or ApicalM chimera plasmids, or no plasmid as a control (Mock). Protein surface expression was analyzed using anti-hSR-B1 (“αH”, blue histograms) and anti-mSR-B1 (“αM”, orange histograms), 24 h after transfection. The grey histogram represents untransfected cells stained with the cognate antibody. (E) CD81KOH16 cells were transfected with hSR-B1, mSR-B1, ApicalH or ApicalM constructs, or no plasmid as a control (Mock), and infected with PbGFP sporozoites 24 h after transfection. The number of infected cells (EEFs) was determined by microscopy after UIS4 staining, 24 h after sporozoite addition (mean control values for each experiment: 59, 139, 214, 245, 299, 315 and 383 EEFs/well in hSR-B1-transfected CD81KOH16 cells). ns, non-significant; *** p < 0.001 (one-way ANOVA followed by Tukey’s multiple comparisons test).

Techniques: Infection, Construct, Transfection, Plasmid Preparation, Control, Expressing, Staining, Microscopy

A key domain within the SR-B1 apex regulates P. berghei infection. (A) Mouse and human protein sequence alignment of the apical region AA 132–223 with the corresponding predicted human secondary structure (alpha helices in red and beta strand in blue). Identical, similar and different amino acids are represented in black, blue and red respectively. Short domains D1, D2 and D3 are delimited by boxes. (B) Predicted tertiary structure of D1, D2 and D3 chimeras by homology modeling, highlighting the segments of mouse (orange) or human (blue) origins. (C) Top views of the electrostatic surface potential of the D1, D2 and D3 chimera apices. Values are in units of kT/e at 298 K, on a scale of − 2 kT/e (red) to + 2 kT/e (blue). White color indicates a neutral potential. Black circles highlight differential electrostatic surface potentials between the different chimeric constructs at the top of the “apex” region. (D) CD81KOH16 cells were transfected with hSR-B1, mSR-B1, D1, D2, or D3 chimeric constructs. Protein surface expression was analyzed using anti-hSR-B1 (“αH”, blue histograms) and anti-mSR-B1 (“αM”, orange histograms), 24 h after transfection. The grey histogram represents untransfected cells stained with the cognate antibody. (E) CD81KOH16 cells were transfected with hSR-B1, mSR-B1, D1, D2, or D3 chimeric constructs, or no plasmid as a control (Mock), and then infected with PbGFP sporozoites 24 h after transfection. The number of infected cells (EEFs) was determined by microscopy after UIS4 staining, 24 h after sporozoite addition (mean control values for each experiment: 59, 139, 214, 245, 299, 315 and 383 EEFs/well in hSR-B1-transfected CD81KOH16 cells). ns, non-significant; ** p < 0.01 (one-way ANOVA followed by Tukey’s multiple comparisons test).

Journal: Scientific Reports

Article Title: Molecular determinants of SR-B1-dependent Plasmodium sporozoite entry into hepatocytes

doi: 10.1038/s41598-020-70468-2

Figure Lengend Snippet: A key domain within the SR-B1 apex regulates P. berghei infection. (A) Mouse and human protein sequence alignment of the apical region AA 132–223 with the corresponding predicted human secondary structure (alpha helices in red and beta strand in blue). Identical, similar and different amino acids are represented in black, blue and red respectively. Short domains D1, D2 and D3 are delimited by boxes. (B) Predicted tertiary structure of D1, D2 and D3 chimeras by homology modeling, highlighting the segments of mouse (orange) or human (blue) origins. (C) Top views of the electrostatic surface potential of the D1, D2 and D3 chimera apices. Values are in units of kT/e at 298 K, on a scale of − 2 kT/e (red) to + 2 kT/e (blue). White color indicates a neutral potential. Black circles highlight differential electrostatic surface potentials between the different chimeric constructs at the top of the “apex” region. (D) CD81KOH16 cells were transfected with hSR-B1, mSR-B1, D1, D2, or D3 chimeric constructs. Protein surface expression was analyzed using anti-hSR-B1 (“αH”, blue histograms) and anti-mSR-B1 (“αM”, orange histograms), 24 h after transfection. The grey histogram represents untransfected cells stained with the cognate antibody. (E) CD81KOH16 cells were transfected with hSR-B1, mSR-B1, D1, D2, or D3 chimeric constructs, or no plasmid as a control (Mock), and then infected with PbGFP sporozoites 24 h after transfection. The number of infected cells (EEFs) was determined by microscopy after UIS4 staining, 24 h after sporozoite addition (mean control values for each experiment: 59, 139, 214, 245, 299, 315 and 383 EEFs/well in hSR-B1-transfected CD81KOH16 cells). ns, non-significant; ** p < 0.01 (one-way ANOVA followed by Tukey’s multiple comparisons test).

Techniques: Infection, Sequencing, Construct, Transfection, Expressing, Staining, Plasmid Preparation, Control, Microscopy

Review

Structured Review

Images

Similar Products

Image Search Results