Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Hepatitis C virus utilizes VLDLR as a novel entry pathway

doi: 10.1073/pnas.1506524113

Figure Lengend Snippet: Effect of HCV E2 and ApoE on VLDLR-mediated HCV entry. (A) Huh7.5 or Huh7 cells transfected with a VLDLR-expressing plasmid were preincubated with IgG as a control or with anti-VLDLR for 1 h at 37 °C. (B and C) After treatment, the cells were infected with Luc-HCVccJFH1 (MOI = 0.1) for 48 h (average ± SD; n = 3). Luc-HCVccJFH1 was preincubated with an IgG control or anti-ApoE (B) and anti-HCV E2 (C). Huh7.5 or Huh7 cells transfected with a VLDLR-expressing plasmid were infected with antibody-treated Luc-HCVccJFH1 (MOI = 0.1) for 48 h (average ± SD; n = 3). (D) Recombinant VLDLR-coated plates were reacted with purified HCV E2 or with purified HCV E2 treated with anti-E2 antibody. The signal was detected using an anti-FLAG antibody and HRP-conjugated mouse IgG. Light absorbance was measured at 450 nm (average ± SD; n = 3). The data shown represent three independent experiments. *P < 0.05, ***P < 0.005 (Student’s t-test).

Article Snippet: Recombinant human VLDLR was purchased from Sino Biological.

Techniques: Transfection, Expressing, Plasmid Preparation, Infection, Recombinant, Purification

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Hepatitis C virus utilizes VLDLR as a novel entry pathway

doi: 10.1073/pnas.1506524113

Figure Lengend Snippet: Mouse VLDLR promotes HCV entry. (A) VLDLR expression was analyzed by RT-PCR using cDNAs from human and mouse liver tissue. G3PDH was used as an internal control. (B) Huh7.5 #26 cells transfected with empty or mouse VLDLR-expressing plasmid were infected with HCVccJFH1 (MOI = 1). The cells were stained with NS5A (red) and VLDLR (6A6) (green) antibodies 48 h postinfection. The images were analyzed by confocal microscopy. (Scale bars, 20 μm.) (C) Hepa1-6 cells (mouse hepatocytes) transfected with an empty vector, a human VLDLR-expressing plasmid, or a mouse VLDLR-expressing plasmid were infected with Luc-VSV-Gpp or Luc-HCVpp (2a). The luciferase activity was analyzed 72 h postinfection (average ± SD; n = 3). (D) Human and mouse VLDLR expression in Hepa1-6 cells was assessed by immunoblotting. The data shown represent three independent experiments. ***P < 0.005 (Student’s t-test).

Article Snippet: Recombinant human VLDLR was purchased from Sino Biological.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Transfection, Plasmid Preparation, Infection, Staining, Confocal Microscopy, Luciferase, Activity Assay, Western Blot

Journal: Journal of Extracellular Vesicles

Article Title: Lipid‐based strategies used to identify extracellular vesicles in flow cytometry can be confounded by lipoproteins: Evaluations of annexin V, lactadherin, and detergent lysis

doi: 10.1002/jev2.12200

Figure Lengend Snippet: Staining of ApoB‐containing lipoproteins with PE‐conjugated polyclonal anti‐ApoB‐48/100 antibody. (a) Scatter plots depicting large‐angle light scatter versus ApoB‐PE fluorescence of commercial VLDL pre‐diluted 8‐fold and stained with fluorescent anti‐ApoB antibody compared to matched isotype, unstained and buffer with reagent controls. (b) Scatter plots depicting large‐angle light scatter versus ApoB‐PE fluorescence of commercial chylomicrons pre‐diluted 4‐fold and stained with fluorescent anti‐ApoB antibody compared to isotype and unstained controls. (c) Scatter plots depicting large‐angle light scatter versus ApoB‐PE fluorescence of platelet poor plasma (PPP) pre‐diluted 32‐fold and stained with fluorescent anti‐ApoB antibody compared to isotype and unstained controls. Light scatter and fluorescence intensities in (A) and (B) are denoted in arbitrary units. (d) Serial dilution control of stained PPP presented with a linear relationship between ApoB event concentration not corrected for dilution factor (left y‐axis, black) and reciprocal dilution factor ( x ‐axis), while the median ApoB‐fluorescence for ApoB‐positive particles (right y ‐axis, orange) was stable. Triangles depict the dilution factor used for all subsequent experiments. (e) Light scatter distributions of ApoB‐positive events in commercial VLDL (red) and chylomicrons (blue) overlayed with silica nanospheres (black, dotted) with an assumed similar RI of 1.47 to lipoproteins. Silica nanospheres from left to right: 100 nm; 180 nm; 300 nm; 590 nm; 1300 nm. ApoB: Apolipoprotein B; Chylo: Chylomicrons; LALS: Large‐angle light scatter (side scatter); VLDL: Very‐low density lipoprotein; Ab: Antibody; PPP: Platelet‐poor plasma

Article Snippet: 365‐10) or chylomicrons (Lee Biosolutions; Cat.no.

Techniques: Staining, Fluorescence, Clinical Proteomics, Serial Dilution, Control, Concentration Assay

Journal: Journal of Extracellular Vesicles

Article Title: Lipid‐based strategies used to identify extracellular vesicles in flow cytometry can be confounded by lipoproteins: Evaluations of annexin V, lactadherin, and detergent lysis

doi: 10.1002/jev2.12200

Figure Lengend Snippet: Illustrations of the similarities and differences between the large ApoB‐containing lipoproteins (VLDL and chylomicrons) and EVs. The yellow alpha‐helical structures on the VLDL and chylomicron represent ApoB‐100 and ApoB‐48, respectively. The blue spheres on all particles refer to phospholipids while the red spheres represent PS phospholipids. The additional coloured components on the EV represent different types of membrane proteins such as tetraspannins (green). Cholesterol and other membrane components including other types of apolipoproteins on the lipoproteins have been left out for the sake of simplicity

Article Snippet: 365‐10) or chylomicrons (Lee Biosolutions; Cat.no.

Techniques: Membrane

Journal: bioRxiv

Article Title: Proteomic Profile of TGF-β1 treated Lung Fibroblasts identifies Novel Markers of Activated Fibroblasts in the Silica Exposed Rat Lung

doi: 10.1101/431825

Figure Lengend Snippet: A-C) Western blot analysis for TM214, VLDLR and SDC2 in control and silica exposed rat lung tissues. Quantitative analyses of immunoblots are shown in (C).D) IHC for expression VLDLR and SDC2 in lung tissues from control (left) andsilica-exposed rats. Statistical significance was assessed using the Student’s t-test.

Article Snippet: The samples were then incubated with primary antibodies against collagen V(COL V, A1515, ABclonal Biotech, Wuhan, China), collagen XI (COL XI, DF3553, Affinity Biosciences, Cincinnati, OH, USA), VLDLR (TA309928, OriGene Technologies, Rockville, MD, USA) or SDC2 (A1810, ABclonal Biotech, Wuhan, China) at 4°C overnight.

Techniques: Western Blot, Expressing

Journal: bioRxiv

Article Title: Proteomic Profile of TGF-β1 treated Lung Fibroblasts identifies Novel Markers of Activated Fibroblasts in the Silica Exposed Rat Lung

doi: 10.1101/431825

Figure Lengend Snippet: A) Effectiveness of different siRNA probes in knockdown expression of VLDLR or SDC2. B) siRNA knockdown of VLDLR reduces expression of COL I and α-SMA in TGF-β1 treated MRC-5 fibroblasts. siRNA knockdown of SDC2 reduces expression of COL1 and causes a non-significant reduction in α-SMA in TGF-β1 treated MRC-5 fibroblasts.Statistical significance was assessed using the one way ANOVA.

Article Snippet: The samples were then incubated with primary antibodies against collagen V(COL V, A1515, ABclonal Biotech, Wuhan, China), collagen XI (COL XI, DF3553, Affinity Biosciences, Cincinnati, OH, USA), VLDLR (TA309928, OriGene Technologies, Rockville, MD, USA) or SDC2 (A1810, ABclonal Biotech, Wuhan, China) at 4°C overnight.

Techniques: Expressing