Journal: PLoS Pathogens

Article Title: An Arginine-Rich Motif in the ORF2 capsid protein regulates the hepatitis E virus lifecycle and interactions with the host cell

doi: 10.1371/journal.ppat.1010798

Figure Lengend Snippet: (A) Dose-response inhibition of ORF2g/c secretion in mycolactone-treated cells. At 7 d.p.e., PLC3/HEV-p6 and mock cells were treated for 24h with the indicated concentrations of mycolactone (in nM) or maximal volume of the vehicle, ethanol (indicated as 0 nM). Supernatants (SN) and lysates (Cells) were collected and ORF2 proteins were detected by WB using the 1E6 Ab. Tubulin served as control protein loading. (B) Schematic representation of ORF2i/g/c proteins and recognition sites of P1H1 and P3H2 antibodies used to discriminate the different ORF2 forms. SP, signal peptide. PC, proprotein convertase. Glycans are in black. (C) Immunoprecipitation of ORF2 proteins in SN and lysates of PLC3/HEV-p6 cells by P1H1, P3H2 and isotype control (CTL) antibodies immobilized on magnetic beads. ORF2 proteins were detected by WB using the 1E6 Ab. (D-F) At 7 d.p.e., PLC3/HEV-p6 cells were treated with the indicated concentrations of Decanoyl-RVKR-chloromethylketone (CMK), hexa-D-arginine amide (D6R) and SSM3 trifluoroacetate (SSM3) (in μM) or DMSO diluent (indicated as 0 μM). (G) At 7 d.p.e., PLC3/HEV-p6 and PLC3 mock cells were transfected with siRNA targeting furin (siFur) or non-targeting siRNA (siCTL) or left non-transfected (NT). (D-G) At 72h post-treatment or post-transfection, supernatants (SN) and lysates (Cells) were collected. SN were immunoprecipitated with P1H1 and P3H2 antibodies and ORF2 proteins were detected by WB using the 1E6 Ab. ORF2intra, αV-Integrin (IntαV) and Tubulin (Tub) were detected in cell lysates. In G, furin (Fur) was also detected in cell lysates. αV-pro-integrin (ProintαV) corresponds to the non-maturated αV-integrin. ORF2g* corresponds to the ORF2g immunoprecipitated by the P1H1 Ab. Molecular mass markers are indicated on the right (kDa).

Article Snippet: Leptomycin B (Cell Signaling), Verdinexor (AdooQ Biosciences), Gossypol (Tocris), Mycolactone A/B toxin [ ], Decanoyl-RVKR-chloromethylketone [CMK] (Sigma), hexa-D-arginine amide [D6R] (Sigma) and SSM3 trifluoroacetate (Tocris) were used in this study.

Techniques: Inhibition, Control, Immunoprecipitation, Magnetic Beads, Transfection

Journal: Frontiers in Veterinary Science

Article Title: Development of a Monoclonal Antibody Against Porcine CD163 SRCR5 Domain Which Partially Blocks Infection of PRRSV

doi: 10.3389/fvets.2020.597843

Figure Lengend Snippet: Confirmation of the specificity of mAb 1D7. (A) . 1D7 was used for the analysis of endogenous CD163 expression of porcine AMs and PK cells with western blotting. (B) Immunofluorescence was used to analysis the specific of 1D7 mAb. (C) . Subtype identification of 1D7 with a mouse antibody subtype identification kit.

Article Snippet: The supernatants of hybridoma cells were diluted 1:100, according to the introduction of the mouse monoclonal antibody subtype identification kit (Proteintech).

Techniques: Expressing, Western Blot, Immunofluorescence

Journal: Frontiers in Veterinary Science

Article Title: Development of a Monoclonal Antibody Against Porcine CD163 SRCR5 Domain Which Partially Blocks Infection of PRRSV

doi: 10.3389/fvets.2020.597843

Figure Lengend Snippet: Mapping the epitope of mAb 1D7. (A) A series of truncated SRCR5-6 were constructed to pCold-TF and was successfully expressed in E. coli BL21(DE3) cells. Protein expression was then induced with IPTG. After incubation at 16°C for 12 h, the cells were harvested for SDS-PAGE. Schematic diagram and western blotting results using 1D7 to probe truncated TF-SRCR5-6. (B) A battery of peptides corresponding to amino acids 497–506 were synthesized. Four hundred nano grams peptides were coated to the ELISA plates in order. Indirect ELISA was used to analysis the epitope of 1D7.

Article Snippet: The supernatants of hybridoma cells were diluted 1:100, according to the introduction of the mouse monoclonal antibody subtype identification kit (Proteintech).

Techniques: Construct, Expressing, Incubation, SDS Page, Western Blot, Battery, Synthesized, Enzyme-linked Immunosorbent Assay, Indirect ELISA