Journal: Journal of Virology

Article Title: Chicken Organic Anion-Transporting Polypeptide 1A2, a Novel Avian Hepatitis E Virus (HEV) ORF2-Interacting Protein, Is Involved in Avian HEV Infection

doi: 10.1128/JVI.02205-18

Figure Lengend Snippet: Designation, prediction, expression, and identification of soluble GST-ap237 in bacteria. (A) Amino acid alignments of genotype 3 human and avian HEV truncated capsid proteins. The alignments of capsid proteins from genotype 3 HEV and CaHEV isolates were performed using the Clustal W module of the MegAlign program of Lasergene 7.1 (DNASTAR, Inc.). (B) 3D structure of the CaHEV capsid protein predicted by using SWISS-MODEL and visualized by using CHIMERA software. The backbone of ap237 (aa 313 to 549) is indicated in green in the pentamer (left). The P domain (blue), M domain (dark blue), S domain (purple), and proline-rich hinge (green) of the monomer are highlighted with different colors. (C) SDS-PAGE of GST-ap237 protein produced in E. coli Transetta(DE3) cells. (D) Western blot analysis of the expression of recombinant GST-ap237 using anti-GST antibody. A GST tag expressed by empty vector pGEX-6P-1 was used as a negative control. (E) Western blot analysis of the expression of recombinant GST-ap237 using the 3E8 mAb. The vertical lines in panels D and E indicate marker (M) images; the Western blot results were taken separately and joined together.

Article Snippet: To express GST-ap237 and GST-1A2 ecto in a bacterial system, recombinant plasmids pGEX-6P-1-ap237 and pGEX-6P-1-1A2 ecto were separately transformed into Escherichia coli Transetta(DE3) (TransGen Biotech, Beijing, China).

Techniques: Expressing, Software, SDS Page, Produced, Western Blot, Recombinant, Plasmid Preparation, Negative Control, Marker

Journal: Journal of Virology

Article Title: Chicken Organic Anion-Transporting Polypeptide 1A2, a Novel Avian Hepatitis E Virus (HEV) ORF2-Interacting Protein, Is Involved in Avian HEV Infection

doi: 10.1128/JVI.02205-18

Figure Lengend Snippet: SDS-PAGE analysis of host proteins by a GST pulldown assay with GST-ap237 and identification of OATP1A2 in chicken embryo hepatocytes (CEH). (A) GST pulldown assay. GST protein was used to exclude the proteins that bind with the GST tag, and GST or GST-ap237 without incubation of the chicken liver cell lysate was set as a blank control. The eluted protein complex was resolved by 10% SDS-PAGE, followed by silver staining. Five visible bands specifically pulled by the ap237 protein are indicated by red arrows. (B) Immunofluorescence assay of OATP1A2 protein in CEH using murine anti-GST-1A2ecto sera and TRITC-goat anti-mouse IgG. Bar, 25 μm. (C) Western blot analysis of OATP1A2 protein using murine anti-GST-1A2ecto sera and HRP-goat anti-mouse IgG. WCL, CEH whole-cell lysates; LMH, LMH cell lysates; Cytos, CEH cytosolic proteins; SMPs & MAPs, solubilized membrane and membrane-associated proteins from CEH; MFs, CEH membrane fractions.

Article Snippet: To express GST-ap237 and GST-1A2 ecto in a bacterial system, recombinant plasmids pGEX-6P-1-ap237 and pGEX-6P-1-1A2 ecto were separately transformed into Escherichia coli Transetta(DE3) (TransGen Biotech, Beijing, China).

Techniques: SDS Page, GST Pulldown Assay, Incubation, Silver Staining, Immunofluorescence, Western Blot

Journal: Journal of Virology

Article Title: Chicken Organic Anion-Transporting Polypeptide 1A2, a Novel Avian Hepatitis E Virus (HEV) ORF2-Interacting Protein, Is Involved in Avian HEV Infection

doi: 10.1128/JVI.02205-18

Figure Lengend Snippet: Proteins present in five specific bands observed in the GST-ap237 lane of identified by MS

Article Snippet: To express GST-ap237 and GST-1A2 ecto in a bacterial system, recombinant plasmids pGEX-6P-1-ap237 and pGEX-6P-1-1A2 ecto were separately transformed into Escherichia coli Transetta(DE3) (TransGen Biotech, Beijing, China).

Techniques: Sequencing

Journal: Journal of Virology

Article Title: Chicken Organic Anion-Transporting Polypeptide 1A2, a Novel Avian Hepatitis E Virus (HEV) ORF2-Interacting Protein, Is Involved in Avian HEV Infection

doi: 10.1128/JVI.02205-18

Figure Lengend Snippet: Interaction and colocalization of avian HEV capsid protein and chicken OATP1A2. (A) Immunoprecipitation with protein G-anti-HA mAb. HEK 293T cells were cotransfected with the recombinant plasmids, and the cell lysates obtained at 48 h posttransfection were immunoprecipitated with anti-HA mAb. The cell lysate and protein G-antibody-antigen complexes were detected by Western blot analysis. (B) Immunoprecipitation with protein G-anti-Flag mAb. (C) Colocalization of OATP1A2 with ap237. HEK 293T cells were cotransfected with recombinant plasmids, and cells were then fixed and subjected to indirect immunofluorescence analysis using mouse anti-HA mAb and rabbit anti-Flag polyclonal antibody. The nucleus is indicated by DAPI (blue) staining in the images.

Article Snippet: To express GST-ap237 and GST-1A2 ecto in a bacterial system, recombinant plasmids pGEX-6P-1-ap237 and pGEX-6P-1-1A2 ecto were separately transformed into Escherichia coli Transetta(DE3) (TransGen Biotech, Beijing, China).

Techniques: Immunoprecipitation, Recombinant, Western Blot, Immunofluorescence, Staining

Journal: Journal of Virology

Article Title: Chicken Organic Anion-Transporting Polypeptide 1A2, a Novel Avian Hepatitis E Virus (HEV) ORF2-Interacting Protein, Is Involved in Avian HEV Infection

doi: 10.1128/JVI.02205-18

Figure Lengend Snippet: The OATP1A2 ectodomain directly interacts with ap237. (A) OATP1A2 structure predicted using the Phyre2 Web portal. (B) SDS-PAGE (left) and Western blot analysis (right) of GST-1A2ecto produced in E. coli Transetta(DE3) cells. (C) SDS-PAGE analysis of the expression of ap237 protein without any tag. The purified ap237 protein was treated with loading buffer containing 2-mercaptoethanol and boiled for 10 min (lane 1) and treated with loading buffer without 2-mercaptoethanol (lane 2). (D) GST pulldown assay to test 1A2ecto binding with ap237. Beads conjugated to GST (lane 1) or GST-1A2ecto (lane 3) were incubated with ap237. Incubation of the beads conjugated to GST-1A2ecto with sp239 (lane 2) was used as a control. After washing, proteins eluted from beads were analyzed via SDS-PAGE and then detected by anti-GST antibodies and the 3E8 mAb. (E) Detection of chicken OATP1A2 binding with ap237 by an indirect ELISA. ELISA plates were coated with different concentrations of ap237 and separately incubated with different concentrations of GST or GST-1A2ecto. The binding of proteins was detected by anti-GST antibodies. (F) Evaluation of OATP1A2 binding with different HEV capsid proteins by an indirect ELISA. Plates were coated with the proteins ap237, sp239, r239, sar239, and ker239 (4 μg/well) and incubated with GST or GST-1A2ecto. PRRSV N protein was used as a negative control.

Article Snippet: To express GST-ap237 and GST-1A2 ecto in a bacterial system, recombinant plasmids pGEX-6P-1-ap237 and pGEX-6P-1-1A2 ecto were separately transformed into Escherichia coli Transetta(DE3) (TransGen Biotech, Beijing, China).

Techniques: SDS Page, Western Blot, Produced, Expressing, Purification, GST Pulldown Assay, Binding Assay, Incubation, Indirect ELISA, Enzyme-linked Immunosorbent Assay, Negative Control

Journal: Journal of Virology

Article Title: Chicken Organic Anion-Transporting Polypeptide 1A2, a Novel Avian Hepatitis E Virus (HEV) ORF2-Interacting Protein, Is Involved in Avian HEV Infection

doi: 10.1128/JVI.02205-18

Figure Lengend Snippet: High levels of OATP1A2 expression enhance ap237 binding to LMH cells. (A) Western blot analysis of ap237 binding to the cell lines LMH, LMHGFP, and LMH1A2-GFP transfected with siNCtrl or si1A2-1. The binding of ap237 to cells was analyzed by Western blotting using the 3E8 mAb. (B) Flow cytometry analysis of ap237 binding to LMH, LMHGFP, and LMH1A2-GFP cells transfected with siNCtrl or si1A2-1. After incubation with ap237, cells were dissociated with enzyme-free cell dissociation buffer and fixed with 4% paraformaldehyde for use in the flow cytometry assay. The mAb 3E8 was used to detect ap237.

Article Snippet: To express GST-ap237 and GST-1A2 ecto in a bacterial system, recombinant plasmids pGEX-6P-1-ap237 and pGEX-6P-1-1A2 ecto were separately transformed into Escherichia coli Transetta(DE3) (TransGen Biotech, Beijing, China).

Techniques: Expressing, Binding Assay, Western Blot, Transfection, Flow Cytometry, Incubation

Journal: Journal of Virology

Article Title: Chicken Organic Anion-Transporting Polypeptide 1A2, a Novel Avian Hepatitis E Virus (HEV) ORF2-Interacting Protein, Is Involved in Avian HEV Infection

doi: 10.1128/JVI.02205-18

Figure Lengend Snippet: Inhibition of avian HEV attachment and infection of LMH1A2-GFP and CEH cells. (A) Cytotoxicity analysis of the substrates (or inhibitors) at their maximum working concentrations (50 μM, 50 μM, 10 μM, and 10 μM for CDCA, SC, carvedilol, and imatinib, respectively) for LMH1A2-GFP cells. (B) Detection of mRNA and protein levels of OATP1A2 expression in LMH1A2-GFP cells treated with different substrates or an inhibitor. Anti-GFP antibody was used to detect the OATP1A2 fusions for Western blotting. (C) Inhibition of CaHEV attachment to and infection of LMH1A2-GFP cells by the substrates or inhibitor. After preincubation with CDCA, SC, carvedilol, and imatinib, CaHEV attachment (left) and infection (right) assays were evaluated using LMH1A2-GFP cells. CaHEV ORF3 RNA was quantified by TaqMan real-time RT-PCR. (D) Blocking CaHEV attachment (left) and infection (right) in LMH1A2-GFP cells by ap237. (E) Anti-GST-1A2ecto mouse sera blocked CaHEV attachment to (left) and infection of (right) LMH1A2-GFP cells. sp239 protein and negative serum were used as controls. CaHEV ORF3 RNA was quantified by TaqMan real-time RT-PCR. Error bars indicate the SEM. (F) Inhibition of CaHEV infection of CEH evaluated using imatinib, ap237, and murine anti-GST-1A2ecto along with the corresponding dimethyl sulfoxide (DMSO), sp239, and negative mouse sera (**, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant).

Article Snippet: To express GST-ap237 and GST-1A2 ecto in a bacterial system, recombinant plasmids pGEX-6P-1-ap237 and pGEX-6P-1-1A2 ecto were separately transformed into Escherichia coli Transetta(DE3) (TransGen Biotech, Beijing, China).

Techniques: Inhibition, Infection, Expressing, Western Blot, Quantitative RT-PCR, Blocking Assay