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: Oncology reports

Article Title: Biological characterisation and application of human MTH1 and monoclonal antibody preparation.

doi: 10.3892/or.2018.6942

Figure Lengend Snippet: Figure 4. Purity analysis. (A) Results of 12% SDS‑PAGE after purification by Ni‑NTA affinity chromatography. Lane M, protein maker; lane 1, supernatant of sonicated cells; lane 2, flow‑through collected from the Ni‑NTA column; lane 3, 50 mM imidazole eluent; lane 4, 100 mM imidazole eluent; lane 5, 250 mM imidazole eluent; lane 6, western blot analysis for recombinant hMTH1. The primary antibody used in the western blot analysis was the anti‑His monoclonal antibody. (B) Results of 12% SDS‑PAGE after purification by G‑50 Gel filtration chromatography. Lane M, protein marker; lane 1, the eluent collected by G‑50 Gel filtration chromatography; lane 2, the recombinant protein hMTH1 that was analysed by western blot analysis. The primary antibody was the anti‑His monoclonal antibody. (C) HPLC analysis of rhMTH1. Only one peak was detected. hMTH1, human MutT homolog 1; HPLC, high‑performance liquid chromatography; SDS‑PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; rhMTH1, recombinant hMTH1 protein.

Article Snippet: Mouse monoclonal antibody subtype identification kit was purchased by ProteinTech Group Inc. (Wuhan, China).

Techniques: Purification, Affinity Chromatography, Sonication, Western Blot, Recombinant, Filtration, Chromatography, Marker, High Performance Liquid Chromatography, Polyacrylamide Gel Electrophoresis

Journal: Oncology reports

Article Title: Biological characterisation and application of human MTH1 and monoclonal antibody preparation.

doi: 10.3892/or.2018.6942

Figure Lengend Snippet: Figure 6. Characteristic determination of MTH1 monoclonal antibody (mAb). (A) The titre of the mouse antiserum, detected by indirect ELISA. Mouse 1, 2 and 3 were immunised with rhMTH1, and the negative control was an unimmunised healthy BALB/c mouse. (B) hMTH1 mAb was detected by SDS‑PAGE. Lane M, the protein marker; lane 1, the sample mixed with non‑reduced loading buffer. Lane 2, the monoclonal antibody treated with reduced loading buffer. (C The specificity of hMTH1 mAb was identified by western blot analysis. Lane 1, the recombinant protein hMTH1 was analysed by western blot analysis. The primary antibody was the prepared anti‑hMTH1 mAb. hMTH1, human MutT homolog 1; rhMTH1, recombinant hMTH1 protein.

Article Snippet: Mouse monoclonal antibody subtype identification kit was purchased by ProteinTech Group Inc. (Wuhan, China).

Techniques: Indirect ELISA, Negative Control, Marker, Western Blot, Recombinant

Journal: Oncology reports

Article Title: Biological characterisation and application of human MTH1 and monoclonal antibody preparation.

doi: 10.3892/or.2018.6942

Figure Lengend Snippet: Figure 7. The isotype, affinity and specificity of the hMTH1 monoclonal antibody (mAb) were tested by ELISA. (A) The results of the MTH1 mAb isotyping. The type of heavy chain was IgG2b and the type of light chain was κ. (B) The results of the hMTH1 monoclonal antibody affinity constant determination. Two concentrations (1 and 2 µg/ml) of hMTH1 were utilised in indirect ELISA to calculate the affinity constant, which was 8.73x10‑9 M. (C) The results of the cell ELISA. The MCF‑7 cells were used to confirm the specificity of the hMTH1 antibody against natural hMTH1. The negative control was 1% BSA. hMTH1, human MutT homolog 1.

Article Snippet: Mouse monoclonal antibody subtype identification kit was purchased by ProteinTech Group Inc. (Wuhan, China).

Techniques: Enzyme-linked Immunosorbent Assay, Indirect ELISA, Negative Control

Journal: Oncology reports

Article Title: Biological characterisation and application of human MTH1 and monoclonal antibody preparation.

doi: 10.3892/or.2018.6942

Figure Lengend Snippet: Figure 8. Immunofluorescence of cultured MCF‑7 cells with the MTH1 monoclonal antibody (mAb). Green fluorescent labelling of protein stained with anti‑hMTH1 mAb, blue nuclear staining with DAPI, and merged images of FITC and DAPI are presented. Calibration bar, 100 µm for all images. hMTH1, human MutT homolog 1.

Article Snippet: Mouse monoclonal antibody subtype identification kit was purchased by ProteinTech Group Inc. (Wuhan, China).

Techniques: Immunofluorescence, Cell Culture, Staining