goat anti guinea pig serum 170  (Vector Laboratories)

Journal: Journal of Virology

Article Title: Deletion of both the Tyrosine-Based Endocytosis Signal and the Endoplasmic Reticulum Retrieval Signal in the Cytoplasmic Tail of Spike Protein Attenuates Porcine Epidemic Diarrhea Virus in Pigs

doi: 10.1128/JVI.01758-18

Figure Lengend Snippet: Mutation of the YxxΦ and KVHVQ motifs of PEDV S protein altered the expression of S proteins on the cell surface and syncytium formation. (A) IF staining of S proteins expressed on cell surface, or total (surface and intracellular) S proteins (Total S). Vero cells were fixed by 4% formaldehyde at 12 h after transfection with plasmid DNA. Without permeabilization, surface S proteins were stained with guinea pig antiserum GP17 against PEDV S1 followed by Alexa Fluor 488 (AF488)-conjugated goat anti-guinea pig IgG. Total S proteins were observed under the mCherry channel. Scale bar: 30 μm. (B) The fluorescent intensities of the total S proteins (mCherry) were measured on 50 individual cells of each sample. NS, no statistically significant difference ( P > 0.05). (C) Surface S/total S fluorescent intensity ratios (AF488/mCherry) were calculated based on 50 individual cells of each sample. (D) Syncytia induced by mCherry-tagged WT S proteins and their mutants. At 6 h after transfection with the plasmid DNA, Vero cells were cultured in medium containing trypsin (10 μg/ml) for an additional 6 h. Cells were fixed with methanol and total S proteins were detected by IF staining with the antiserum GP17 as described above. Nuclei were stained with DAPI. Scale bar: 50 μm. (E) Nuclei in each syncytium were counted based on 200 syncytia for each sample. Values in panels B, C, and E are shown in box-whisker plots. The boxes indicate interquartile ranges in different groups, the lines in the boxes represent median values, and the whiskers show the range of a group of values excluding the outliers, which are shown as dots. Groups with statistically significant differences ( P < 0.05) are indicated with different letters; the alphabetical order reflects groups with median values from high to low.

Article Snippet: Then the cells were incubated with secondary antibody AF488-conjugated goat anti-guinea pig IgG serum (Invitrogen, Carlsbad, CA).

Techniques: Mutagenesis, Expressing, Staining, Transfection, Plasmid Preparation, Cell Culture, Whisker Assay

Journal: Journal of Virology

Article Title: Deletion of both the Tyrosine-Based Endocytosis Signal and the Endoplasmic Reticulum Retrieval Signal in the Cytoplasmic Tail of Spike Protein Attenuates Porcine Epidemic Diarrhea Virus in Pigs

doi: 10.1128/JVI.01758-18

Figure Lengend Snippet: IF staining of antibody-S protein uptake assay in Vero cells. At 24 h after transfection with the plasmid DNA, Vero cells were incubated with guinea pig anti-S1 antiserum GP17 at 4°C for 10 min. After washing with PBS three times, one set of cells was cultured at 37°C for 30 min to allow endocytosis. Another set of cells was kept at 4°C for 30 min as the control group. Cells were fixed with 4% formaldehyde without permeabilization. Surface S proteins were stained with mouse anti-S2 monoclonal antibody SD129-5 and goat anti-mouse AF647-conjugated secondary antibodies (red). Then the cells were permeabilized with Triton X-100 and stained with goat anti-guinea pig AF488-conjugated secondary antibodies (green). Nuclei were stained with DAPI (blue). Cells were randomly selected, and images were taken by using a Leica TCS SP6 confocal microscope. Scale bar: 10 μm.

Article Snippet: Then the cells were incubated with secondary antibody AF488-conjugated goat anti-guinea pig IgG serum (Invitrogen, Carlsbad, CA).

Techniques: Staining, Transfection, Plasmid Preparation, Incubation, Cell Culture, Microscopy

Journal: Journal of Virology

Article Title: Deletion of both the Tyrosine-Based Endocytosis Signal and the Endoplasmic Reticulum Retrieval Signal in the Cytoplasmic Tail of Spike Protein Attenuates Porcine Epidemic Diarrhea Virus in Pigs

doi: 10.1128/JVI.01758-18

Figure Lengend Snippet: Quantification of the colocalization of Gp17-bound S proteins and surface S proteins and the ratios of surface S/total S levels in the antibody-S protein uptake assay. (A) Colocalization coefficients between the GP17-bound S proteins and surface S proteins (SD129-5) are shown in . PCC values of the two signals in 20 to 25 individual cells cultured at 4°C or 37°C for 30 min were plotted. (B) Quantification of GP17 signals on the cell surface in the Gp17-S uptake assay. The assay was performed as described in the legend to . To stain the GP17 bound on the cell surface, cells were fixed with 4% formaldehyde without permeabilization. GP17 was probed using AF488-conjugated secondary antibodies (green). The fluorescent intensities of surface (AF488) to total S (mCherry) ratios were measured in 20 to 25 individual cells. Values are plotted in a bar chart, and shown as means ± SDs. Values of the same sample cultured at 4°C and 37°C were analyzed by Student’s t test. NS, P > 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Article Snippet: Then the cells were incubated with secondary antibody AF488-conjugated goat anti-guinea pig IgG serum (Invitrogen, Carlsbad, CA).

Techniques: Cell Culture, Staining

Journal: Journal of Virology

Article Title: Deletion of both the Tyrosine-Based Endocytosis Signal and the Endoplasmic Reticulum Retrieval Signal in the Cytoplasmic Tail of Spike Protein Attenuates Porcine Epidemic Diarrhea Virus in Pigs

doi: 10.1128/JVI.01758-18

Figure Lengend Snippet: Internalized S proteins colocalized with early endosomes. Antibody-S protein uptake assays were performed at 24 hpt for the two wild-type S proteins (WT1 and WT2) and the four mutants, Δ5aa, Δ5aa-A, MK-P10, and MK-P10-V, that formed puncta as shown in . Cells were incubated with guinea pig anti-S1 antiserum GP17 at 4°C for 10 min. After washing with PBS three times, one set of cells were cultured at 37°C for 30 min to allow endocytosis and another set of cells were incubated at 4°C for 30 min. After fixation with methanol, early endosome marker Rab5 proteins were stained with rabbit anti-Rab5 MAb (C8B1) followed by AF647-conjugated goat anti-rabbit antibody (red). GP17-bound S proteins were stained with goat anti-guinea pig AF488-conjugated secondary antibodies (green). (A) IF staining of internalized S protein puncta and early endosome marker Rab5. Arrows indicate the GP17-bound S protein puncta or/and early endosome marker Rab5 that colocalized together. Scale bar: 10 μm. (B) Percentage of GP17-S signals colocalized with Rab5 signals in Vero cells. The MCC values of the two signals in 20 to 25 cells in each sample were measured. Values are shown as means ± SDs. Values of the same sample cultured in 4°C or 37°C were analyzed by Student’s t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: Then the cells were incubated with secondary antibody AF488-conjugated goat anti-guinea pig IgG serum (Invitrogen, Carlsbad, CA).

Techniques: Incubation, Cell Culture, Marker, Staining