mouse monoclonal anti-pedf n antibody sd-2  (MedGen Inc)

Journal: Viruses

Article Title: Structural Basis for Inhibiting Porcine Epidemic Diarrhea Virus Replication with the 3C-Like Protease Inhibitor GC376

doi: 10.3390/v12020240

Figure Lengend Snippet: Structure of GC376 and the inhibitory effect of GC376 on the porcine epidemic diarrhea virus (PEDV) 3CL pro determined by FRET assays. ( A ) GC376 is a dipeptidyl bisulfite adduct salt that showed excellent inhibitory effects on some picornaviruses and coronaviruses in enzymatic or cell-based assays and ( B ) an enzyme (100 nM) and a fluorescent peptide substrate (40 μM) were used in the FRET assay, and the concentration of GC376 ranged from 0.03 μM to 80 μM. The 50% inhibitory concentration (IC 50 ) was calculated using GraphPad Prism5 to assess inhibition ratios at different inhibitor concentrations. The error bars show the S.D. of the results from three replicates.

Article Snippet: After blocking with 5% skimmed milk at room temperature for 3 h, the membranes were incubated with an anti-PEDV nucleocapsid (N) protein monoclonal antibody and an anti-GAPDH monoclonal antibody (Proteintech, Rosemont, IL, USA) for 2 h. Then, the membranes were incubated with an HRP-conjugated secondary antibody (Boster Biological Technology, Pleasanton, CA, USA) for 40 min.

Techniques: Virus, Concentration Assay, Inhibition

Journal: Viruses

Article Title: Structural Basis for Inhibiting Porcine Epidemic Diarrhea Virus Replication with the 3C-Like Protease Inhibitor GC376

doi: 10.3390/v12020240

Figure Lengend Snippet: Data collection and refinement statistics.

Article Snippet: After blocking with 5% skimmed milk at room temperature for 3 h, the membranes were incubated with an anti-PEDV nucleocapsid (N) protein monoclonal antibody and an anti-GAPDH monoclonal antibody (Proteintech, Rosemont, IL, USA) for 2 h. Then, the membranes were incubated with an HRP-conjugated secondary antibody (Boster Biological Technology, Pleasanton, CA, USA) for 40 min.

Techniques: Solvent

Journal: Viruses

Article Title: Structural Basis for Inhibiting Porcine Epidemic Diarrhea Virus Replication with the 3C-Like Protease Inhibitor GC376

doi: 10.3390/v12020240

Figure Lengend Snippet: Antiviral effects of GC376 on the replication of two PEDV strains in cell culture. ( A ) CV777 was added at a multiplicity of infection (MOI) of 0.01 with a series of GC376 concentrations (0.78 μM, 1.56 μM, 3.13 μM, 6.25 μM, 12.5 μM, 25 μM, 50 μM and 100 μM). Cell viability was measured using Celltiter-Glo Luminescent Cell Viability Assay reagent (Promega, Madison, WI, USA) at 36 h. Finally, the 50% maximal effect (EC 50 ) values were calculated using GraphPad Prism 5 to assess inhibition ratios at different inhibitor concentrations. The error bars show the S.D. of the results from three replicates. ( B ) The EC 50 of YN144 was measured as above except that the cell viability was measured at 12h. The error bars show the S.D. of the results from three replicates.

Article Snippet: After blocking with 5% skimmed milk at room temperature for 3 h, the membranes were incubated with an anti-PEDV nucleocapsid (N) protein monoclonal antibody and an anti-GAPDH monoclonal antibody (Proteintech, Rosemont, IL, USA) for 2 h. Then, the membranes were incubated with an HRP-conjugated secondary antibody (Boster Biological Technology, Pleasanton, CA, USA) for 40 min.

Techniques: Cell Culture, Infection, Cell Viability Assay, Inhibition

Journal: Viruses

Article Title: Structural Basis for Inhibiting Porcine Epidemic Diarrhea Virus Replication with the 3C-Like Protease Inhibitor GC376

doi: 10.3390/v12020240

Figure Lengend Snippet: Western blot analysis of the effects of GC376 on the replication of two PEDV strains in cell culture. ( A , B ) Vero cells were infected with one of two PEDV strains at an MOI of 0.01 and then treated with GC376 at different concentrations. Cell extracts were analyzed by western blotting for nucleocapsid expression, and GAPDH was used as an internal control. ( C ) Indirect immunofluorescence assays are shown. The white bar measure is 400 μm. YN144 was used to infect cells at an MOI of 0.01, and GC376 was added to the cells at a series of concentrations. The cells were treated with primary antibody (anti PEDV N) and then a FITC-labeled secondary antibody. The cells infected by viruses were captured in green as shown in the picture.

Article Snippet: After blocking with 5% skimmed milk at room temperature for 3 h, the membranes were incubated with an anti-PEDV nucleocapsid (N) protein monoclonal antibody and an anti-GAPDH monoclonal antibody (Proteintech, Rosemont, IL, USA) for 2 h. Then, the membranes were incubated with an HRP-conjugated secondary antibody (Boster Biological Technology, Pleasanton, CA, USA) for 40 min.

Techniques: Western Blot, Cell Culture, Infection, Expressing, Control, Immunofluorescence, Labeling

Journal: Viruses

Article Title: Structural Basis for Inhibiting Porcine Epidemic Diarrhea Virus Replication with the 3C-Like Protease Inhibitor GC376

doi: 10.3390/v12020240

Figure Lengend Snippet: Crystal structure of the PEDV 3CL pro in complex with GC376. ( A ) Electron density map of GC376 (2Fo-Fc, contoured at 1.0 σ). The bisulfite group of GC376 was removed, and the compound formed a covalent bond with Cys144; ( B ) three-dimensional structure of the substrate-binding pockets occupied by GC376. The pocket is shown as the surface, and the two residues Thr 47 and Asn 141 are shown as sticks. The distance between the two residues is labeled. GC376 is shown in yellow as sticks. The glutamine surrogate ring and the leucine of GC376 fit comfortably into the S1 and S2 binding sites, respectively. The 6-membered aromatic ring of GC376 points to the solvent; ( C ) diagram of the detailed molecular interactions between GC376 and the protease. The residues of the pockets are represented in green as lines, and GC376 is shown in yellow as sticks. Hydrogen bond interactions are shown as black dashed lines; ( D ) structural diagram of the distributions of hydrophobic and hydrophilic interactions at the interface of the PEDV 3CL pro in complex with GC376. The residues of the protease are shown as orange sticks and red arcs with spokes. GC376 is shown in blue as sticks. Carbon, nitrogen, oxygen and sulfur atoms are shown as black, blue, red and yellow circles, respectively. Hydrogen bonds are shown as green dashed lines labeled with the distance between the donor atom and corresponding acceptor atom. Hydrophobic interactions are demonstrated by arcs with spokes radiating toward the atoms (with spokes around) or residues (shown as arcs with spokes) they contact.

Article Snippet: After blocking with 5% skimmed milk at room temperature for 3 h, the membranes were incubated with an anti-PEDV nucleocapsid (N) protein monoclonal antibody and an anti-GAPDH monoclonal antibody (Proteintech, Rosemont, IL, USA) for 2 h. Then, the membranes were incubated with an HRP-conjugated secondary antibody (Boster Biological Technology, Pleasanton, CA, USA) for 40 min.

Techniques: Binding Assay, Labeling, Solvent

Journal: Viruses

Article Title: Structural Basis for Inhibiting Porcine Epidemic Diarrhea Virus Replication with the 3C-Like Protease Inhibitor GC376

doi: 10.3390/v12020240

Figure Lengend Snippet: Substrate specificity of the PEDV 3CL pro at the P2 site and the optimization of GC376. ( A ) The structure of PEDV 3CL pro -GC376 (light magenta) is superimposed over the complex structure of PEDV 3CL pro -peptide (yellow, PDB: 4ZUH). GC376 and the peptide substrate are shown as sticks with balls. The P1, P2 and P3 residues of the substrate and the Leu residue of GC376 are shown and labeled; ( B ) an enzyme (100 nM) and fluorescent peptide substrates (40 μM) were used in the FRET assay. The relative fluorescence units (RFU) detected upon cleavage of the fluorescent peptide substrate and its seven variants by the PEDV 3CL pro are shown in the figure; ( C , D ) viability of YN144-infected Vero cells after treatment with the two GC376 variants, Target 1 (L-M) and Target 2 (L-F), was measured using the Celltiter-Glo Luminescent Cell Viability Assay reagent (Promega, Madison, WI, USA). EC 50 values were calculated using GraphPad Prism5 to assess inhibition ratios at different inhibitor concentrations. The structures of the two compounds are shown in the figure, and their modification sites are labeled. All the error bars show the S.D. of the results from three replicates.

Article Snippet: After blocking with 5% skimmed milk at room temperature for 3 h, the membranes were incubated with an anti-PEDV nucleocapsid (N) protein monoclonal antibody and an anti-GAPDH monoclonal antibody (Proteintech, Rosemont, IL, USA) for 2 h. Then, the membranes were incubated with an HRP-conjugated secondary antibody (Boster Biological Technology, Pleasanton, CA, USA) for 40 min.

Techniques: Residue, Labeling, Fluorescence, Infection, Cell Viability Assay, Inhibition, Modification

Journal: Viruses

Article Title: Structural Basis for Inhibiting Porcine Epidemic Diarrhea Virus Replication with the 3C-Like Protease Inhibitor GC376

doi: 10.3390/v12020240

Figure Lengend Snippet: Structure alignment between the PEDV 3CL pro –GC376 and transmissible gastroenteritis virus (TGEV) 3CL pro –GC376. ( A ) The structure of PEDV 3CL pro –GC376 (red) is superimposed with the complex structure of TGEV 3CL pro –GC376 (blue). GC376 is shown as sticks with balls, and the residues of 3CL pro involved in binding with GC376 are labeled and shown as lines. The conserved residues between the two 3CL pro s are labeled in black and nonconserved residues are labeled in different colors. The black arrows indicate the structural deviation of the Leu side chain and 6-membered aromatic ring of GC376 between the two structures and ( B ) the root mean square deviation (RMSD) values for the residues involved in the GC376 binding of the two structures.

Article Snippet: After blocking with 5% skimmed milk at room temperature for 3 h, the membranes were incubated with an anti-PEDV nucleocapsid (N) protein monoclonal antibody and an anti-GAPDH monoclonal antibody (Proteintech, Rosemont, IL, USA) for 2 h. Then, the membranes were incubated with an HRP-conjugated secondary antibody (Boster Biological Technology, Pleasanton, CA, USA) for 40 min.

Techniques: Virus, Binding Assay, Labeling