v5-specific mab a00641 antibody  (GenScript corporation)

Journal: The Journal of General Virology

Article Title: Molecular cloning of porcine Siglec-3, Siglec-5 and Siglec-10, and identification of Siglec-10 as an alternative receptor for porcine reproductive and respiratory syndrome virus (PRRSV)

doi: 10.1099/jgv.0.000859

Figure Lengend Snippet: Expression analysis of Siglec-3, Siglec-5 and Siglec-10 using IFA and Western blotting. (a) PK-15 cells were transfected with Siglec-3-, Siglec-5- and Siglec-10-encoding vector. Twenty-four hours post-transfection, the cells were fixed with 4 % PF and permeabilized with Triton X-100 for cytoplasmic staining, or not permeabilized for surface staining. IFA was performed using V5-specific antibody (Siglec; green) and Hoechst 33 342 (nuclei; blue). Scale bar: 25 µm (b) Western blot identification. HEK-293T cells were transfected with Siglec-3, Siglec-5 and Siglec-10. Twenty-four hours post-transfection, the cells were collected and lysed with lysis buffer. Afterwards, cellular lysates were treated or mock-treated with sialidase, EndoH or PNGase F and analysed by SDS-PAGE and Western blot. Primary antibody V5-specific mAb (GenScript; A00641) and secondary peroxidase-labelled goat anti-mouse IgG antibodies (Dako) were used for immunoblotting. For the detection of tubulin, an HRP-conjugated anti-alpha tubulin monoclonal antibody (Abcam; ab40742) was used.

Article Snippet: A V5-specific mAb (GenScript; A00641) diluted 1 : 2000 in PBS and peroxidase-labelled goat anti-mouse IgG antibodies (Dako) diluted 1 : 1000 in blocking solution were used for the detection of protein.

Techniques: Expressing, Western Blot, Transfection, Plasmid Preparation, Staining, Lysis, SDS Page

Journal: The Journal of General Virology

Article Title: Molecular cloning of porcine Siglec-3, Siglec-5 and Siglec-10, and identification of Siglec-10 as an alternative receptor for porcine reproductive and respiratory syndrome virus (PRRSV)

doi: 10.1099/jgv.0.000859

Figure Lengend Snippet: Virus production for the different transfected PK-15 cell groups 24 h after infection. PK-15 cells were transiently transfected with a pCD163-encoding vector in combination with a Siglec-1, Siglec-3, Siglec-5, Siglec-10 or control vector, and 48 h after transfection the cells were treated or not treated with sialidase for 1 h and inoculated with PRRSV LV or MN-184 for 1 h. Twenty-four hours post-infection, the cells were fixed and stained for infection and expression of the different Siglecs and CD163. (a) Immunofluorescence staining of infected cells with mAb 13E2 (against PRRSV nucleocapsid protein; green) and Hoechst 33 342 (nuclei; blue). The absolute number of infected cells for each condition was determined and displayed in the images as the mean ± SEM of three independent experiments. Scale bar: 50 µm. (b) Expression analysis of the different Siglecs using fluorescence microscopy. PK-15 cells were fixed, permeabilized and stained with V5-specific mAb (green) and Hoechst 33 342 (nuclei; blue). The absolute number of transfected cells was determined for each condition and is displayed in the images as the mean ± SEM of three independent experiments. Scale bar: 50 µm. (c) To evaluate virus production, the cell supernatants collected at 24 h p.i. were titrated and the results are displayed in the graphs. The CD163/Siglec double-transfected groups that were significantly different from the CD163 single-transfected group are represented as * P <0.05; ** P <0.01 and *** P <0.001.

Article Snippet: A V5-specific mAb (GenScript; A00641) diluted 1 : 2000 in PBS and peroxidase-labelled goat anti-mouse IgG antibodies (Dako) diluted 1 : 1000 in blocking solution were used for the detection of protein.

Techniques: Virus, Transfection, Infection, Plasmid Preparation, Control, Staining, Expressing, Immunofluorescence, Fluorescence, Microscopy