Journal: Gene therapy

Article Title: Replicating reoviruses with a transgene replacing the codons for the head domain of the viral spike.

doi: 10.1038/gt.2014.126

Figure Lengend Snippet: Figure 4. Detection of σ1-His in infected 911scFvHis cells and CsCl-purified particles. (a) The 911scFvHis cells were infected with early passage rS1His-2A-iLOV. Lysates of infected cells were made 24 or 48 h postinfection (h.p.i) and analyzed by western blotting. As a positive control for σ1-His protein, cells transfected with pCMVS1His-2A-iLOV are used and lysates were made 48 h.p.i. Cells infected with wt reovirus (24 h.p.i) are taken along to visualize the difference in size; predicted size of wt-σ1 is about 49 kDa and σ1-His after P2A processing is about 30 kDa; σ1 is detected with S1R3, a polyclonal rabbit antibody used against N-term of σ1. As a loading control, a β-actin antibody is used. (b) Approximately 5 × 109 particles of CsCl-purified wt reovirus or σ1-His containing rS1His-2A-iLOV reovirus was loaded onto 12% SDS-PAGE gels. S1R3 polyclonal antibody against N-term of σ1 was used for the detection of both the wt-σ1 and the σ1-His protein (arrow). The smaller σ1-His protein is also detected with an antibody used against the P2A peptide. The dot indicates a background band, probably crossreactivity with σ3. As an indication of the amount of particles that are loaded on the gel, σ3 is detected with the monoclonal antibody 4F2.

Article Snippet: Mouse monoclonal antibody 4F2, used against reovirus σ3 obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biology (Iowa City, IA, USA).44 Antibody (mouse) used against β-actin: ImmunO anti-Actin clone C4 (MP Biomedicals, Eindhoven, The Netherlands).

Techniques: Infection, Western Blot, Positive Control, Transfection, Control, SDS Page