recombinant human netrin 1 his  (R&D Systems)

Journal: PLOS Pathogens

Article Title: Netrin-1 inhibits the attachment and internalization of Hepatitis B virus for hepatocyte infection

doi: 10.1371/journal.ppat.1013776

Figure Lengend Snippet: A: Schematic of HBV attachment to hepatocytes. B: Dox-inducible Netrin-1-overexpressing HepG2-NTCP-YFP cells were incubated with or without 25 ng/mL Dox for 72 h and then inoculated with the preS1-probe at 4°C for 1.5 h to allow surface attachment (upper). After fixation, fluorescence was observed by confocal microscopy. Representative images (preS1-probe, red; NTCP-YFP, green; nucleus, blue), shown at low (scale bar, 10 µm) and high (scale bar, 4 µm) magnification (lower). C: Dox-inducible Netrin-1-overexpressing HepG2-NTCP-YFP cells were incubated with 0, 1, 5, or 25 ng/mL Dox for 72 h to induce Netrin-1 expression. The cells were then pretreated with heparanase (0.5 µg/mL) at 37°C for 1.5 h to remove HSPG heparan sulfate side chains, followed by incubation with HBV at 4°C for 1.5 h. Surface-bound HBV DNA was assessed by qPCR. D: Structure of wild-type and truncated Netrin-1 variants. LE1, LE2, and LE3, laminin-type epidermal growth factor-like repeats; LN, laminin domain; NTR, netrin-like domain. E: HepG2 cells were transfected with Netrin-1-Flag and LIPG-HA (upper) or HBs-Flag and Netrin-1-HA (lower) plasmids. At 48 h after transfection, the cells were immunoprecipitated with α-Flag antibody (for Netrin-1) or α-HA antibody (for LIPG) and detected using α-HA antibody (for LIPG) or α-Flag antibody (for Netrin-1) (upper), immunoprecipitated with α-Flag antibody (for HBs) or α-HA antibody (for Netrin-1) and detected by α-HA antibody (for Netrin-1) or α-Flag antibody (for HBs) (lower). F: HepG2 cells were co-transfected with LIPG-V5 and wild-type or truncated Netrin-1-HA plasmids. At 48 h after transfection, the cells were immunoprecipitated with α-V5 antibody (for LIPG) or α-HA antibody (for Netrin-1) and detected by α-V5 antibody (for LIPG) or α-HA antibody (for Netrin-1). G and H: Dox-inducible HepG2-NTCP-YFP cells expressing wild-type or truncated Netrin-1 were cultured with or without 25 ng/mL Dox for 72 h, followed by detection. Culture supernatants, membrane fractions, and whole cell lysates were subjected to western blotting analysis (G), and an HBV attachment assay was performed (H). Data are the mean ± standard error of the mean ( n = 3). Statistical analysis was performed using two-way ANOVA with Tukey’s test. **** p < 0.0001, *** p < 0.001, * p < 0.05, ns = not significant.

Article Snippet: Open reading frames of the wild-type and truncated forms of Netrin-1 and LIPG (with the signal sequence retained to ensure proper secretion) were amplified using the Netrin-1 cDNA plasmid (RC218429; OriGene, Rockville, MD) or LIPG cDNA plasmid (RC209248; OriGene) as templates.

Techniques: Incubation, Fluorescence, Confocal Microscopy, Expressing, Transfection, Immunoprecipitation, Cell Culture, Membrane, Western Blot

Journal: PLOS Pathogens

Article Title: Netrin-1 inhibits the attachment and internalization of Hepatitis B virus for hepatocyte infection

doi: 10.1371/journal.ppat.1013776

Figure Lengend Snippet: A and B: Schematic representation of the heparin/heparan sulfate-binding sites of Netrin-1 and the substituted amino acid mutations. C: Diagram of wild-type LIPG and cleaved NTD and CTD, with or without deletion of the heparin-binding motif. D and E: HepG2 cells were co-transfected with a series of truncated forms of LIPG-HA and wild-type Netrin-1 V domain (V-Flag) (D) or heparin/heparan sulfate-binding site mutant V domain (V mut-V5) (E) plasmids. The cells were immunoprecipitated with α-HA antibody (for LIPG) and then detected with α-Flag antibody (for V) or α-V5 antibody (for V mut). F and G: HepG2 cells were co-transfected with a series of truncated forms of LIPG-HA and wild-type Netrin-1 C domain (C-Flag) (F) or heparin/heparan sulfate-binding site mutant C domain (C mut-V5) (G) plasmids. Cells were immunoprecipitated with α-HA antibody (for LIPG) and then detected with α-Flag antibody (for C) or α-V5 antibody (for C mut). H: A model of the Netrin-1 V domain interacting with LIPG. The Netrin-1 V domain interacts with the NTD heparin-binding motif of LIPG, while the heparin/heparan sulfate-binding site mutant V domain loses this ability. I: A model of the Netrin-1 C domain interacting with LIPG. The Netrin-1 C domain interacts with all domains of LIPG, while the heparin/heparan sulfate-binding site mutant C domain loses its affinity for the CTD heparin-binding motif of LIPG, but retains its affinity for the other domains. J: Dox-inducible full-length LIPG-HA-expressing HepG2-NTCP-YFP cells were co-transfected with full-length wild-type Netrin-1-Flag, mutant Netrin-1-V5 with heparin/heparan sulfate-binding site mutation in the V domain, and pcDNA3.1(+) empty plasmids (for a total of 4,250 ng plasmid per sample). The cells were incubated with 25 ng/mL Dox to induce LIPG expression. At 48 h after transfection, the cells were immunoprecipitated with α-HA antibody (for LIPG) and then detected with α-HA antibody (for LIPG), α-Flag antibody (for wild-type Netrin-1), or α-V5 antibody (for Netrin-1 with mutation in the V domain). K: Dox-inducible full-length LIPG-HA-expressing HepG2-NTCP-YFP cells were co-transfected with full-length wild-type Netrin-1-Flag, mutant Netrin-1-V5 with heparin/heparan sulfate-binding site mutation in the C domain, and pcDNA3.1(+) empty plasmids (for a total of 4,250 ng plasmid per sample). The cells were incubated with 25 ng/mL Dox to induce LIPG expression. At 48 h after transfection, the cells were immunoprecipitated with α-HA antibody (for LIPG) and then detected with α-HA antibody (for LIPG), α-Flag antibody (for wild-type Netrin-1), or α-V5 antibody (for Netrin-1 with mutation in the C domain).

Article Snippet: Open reading frames of the wild-type and truncated forms of Netrin-1 and LIPG (with the signal sequence retained to ensure proper secretion) were amplified using the Netrin-1 cDNA plasmid (RC218429; OriGene, Rockville, MD) or LIPG cDNA plasmid (RC209248; OriGene) as templates.

Techniques: Binding Assay, Transfection, Mutagenesis, Immunoprecipitation, Expressing, Plasmid Preparation, Incubation

Journal: PLOS Pathogens

Article Title: Netrin-1 inhibits the attachment and internalization of Hepatitis B virus for hepatocyte infection

doi: 10.1371/journal.ppat.1013776

Figure Lengend Snippet: A: Proposed mechanism for the binding of LHBs to the C-terminal heparin-binding motif of LIPG CTD. B: HepG2 cells were transfected with LHBs-Flag and LIPG NTD-HA (upper) or LHBs-Flag and LIPG CTD-HA (lower) plasmids. At 48 h after transfection, the cells were immunoprecipitated with α-HA antibody (for LIPG NTD/CTD) or α-Flag antibody (for LHBs) and detected using an α-HA antibody (for LIPG NTD/CTD) or α-Flag antibody (for LHBs). C: HepG2 cells were co-transfected with LHBs-Flag and truncated LIPG CTD-HA plasmids, either containing or lacking the heparin-binding motif. At 48 h after transfection, the cells were immunoprecipitated with α-Flag antibody (for LHBs) or α-HA antibody (for LIPG CTD) and then detected with α-Flag antibody (for LHBs) or α-HA antibody (for LIPG CTD). D: The C-terminal HBS of LIPG (amino acids 331–340; NSKMYLKTRA) was biotinylated at the N-terminus. For each assay, 150 μL of 0.5 mg/mL biotinylated peptide was incubated with a streptavidin-immobilized gel. Subsequently, 100 μL of 1 ng/μL recombinant LHBs, in the presence of 0, 1, 2, or 4 ng/μL recombinant Netrin-1 (rNetrin-1)-His, was added to the peptide-immobilized gel. After incubation, proteins bound to the biotinylated peptide were eluted and subjected to western blotting analysis. rNetrin-1 was detected using α-His antibody, and large HBsAg was detected using α-HBsAg antibody. E: HepG2 cells were co-transfected with LHBs-Flag and wild-type LIPG-V5 plasmids, with or without the addition of wild-type Netrin-1-HA plasmids (lower and upper, respectively). At 48 h after transfection, the cells were immunoprecipitated with α-V5 antibody (for LIPG) and then detected with α-Flag antibody (for LHBs), α-V5 antibody (for LIPG), or α-HA antibody (for Netrin-1). F and G: Dox-inducible HepG2-NTCP-YFP cells expressing wild-type or truncated forms of Netrin-1-HA were co-transfected with LHBs-Flag and wild-type LIPG-V5 expression plasmids, and then incubated with 0, 1, 5, or 25 ng/mL Dox for 48 h. The cells were harvested for immunoprecipitation with α-V5 (for LIPG) or IgG-Ctrl antibody and then detected with α-Flag antibody (for LHBs), α-HA antibody (for Netrin-1), or α-V5 antibody (for LIPG). H: Illustration of wild-type Netrin-1 and Netrin-1 with mutations in the heparin/heparan sulfate-binding sites. I: Dox-inducible HepG2-NTCP-YFP cells expressing wild-type Netrin-1 or heparin-binding-deficient mutant were incubated with 0, 1, 5, or 25 ng/mL Dox for 48 h, and then subjected to the HBV attachment assay. J: Whole cell lysates or isolated membrane fractions from samples under the same conditions as in panel H were subjected to western blotting analysis (upper). The membrane protein Na ⁺ /K ⁺ -ATPase α1 (ATP1A1) was used as a loading control for the membrane fraction. Band intensities of Netrin-1 and LIPG in the membrane fraction were normalized to ATP1A1 and plotted in a graph (lower). K: Depiction of Netrin-1 interfering with the LIPG-dependent enhancement of HBV attachment. (a) HBV binds to the C-terminal heparin-binding motif of LIPG through LHBs, and is then transported to HSPGs via the LIPG-HSPG interaction. Netrin-1 interferes with this process in three ways: (b) it binds to the heparan sulfate side chains of HSPGs, causing the release of LIPG from HSPGs; (c) Netrin-1 interacts with free LIPG, occupying its heparin-binding motif and preventing LIPG from anchoring to HSPGs; and (d) Netrin-1 competes with LHBs for binding to the C-terminal heparin-binding motif of LIPG. Data are the mean ± standard error of the mean ( n = 3). Statistical analysis was performed using two-way ANOVA with Tukey’s test. **** p < 0.0001, *** p < 0.001, * p < 0.05, ns = not significant.

Article Snippet: Open reading frames of the wild-type and truncated forms of Netrin-1 and LIPG (with the signal sequence retained to ensure proper secretion) were amplified using the Netrin-1 cDNA plasmid (RC218429; OriGene, Rockville, MD) or LIPG cDNA plasmid (RC209248; OriGene) as templates.

Techniques: Binding Assay, Transfection, Immunoprecipitation, Incubation, Recombinant, Western Blot, Expressing, Mutagenesis, Isolation, Membrane, Control