Fig. 3: CPMV viral protein detection in mammalian cells.

a Western blot detecting CPMV coat proteins in RAW 264.7 macrophage cells at timepoint 0 h, 24 h, 48 h, 72 h, 96 h after CPMV was incubated with RAW 264.7 macrophage cells for 24 h. L-CP and S-CP are detectable at 0 h, to a lesser degree at 24 h. GAPDH staining confirms equal concentration loading of protein lysate. Controls are negative for CPMV detection. Non-specific staining is observed at ~47 kDa. b Fluorescently labeled CPMV-Cy5 was incubated with RAW 264.7 murine macrophage cells and flow cytometry was used to measure CPMV levels by means of Cy5 fluorescence. Median fluorescent intensity (MFI) shows CPMV-Cy5 at 0 h time point; signals decrease over time. N = 2. c No detection of CPMV 24 K protease in RAW 264.7 macrophage cells incubated with CPMV; CPMV-infected plant sap served as positive control. This data indicates that de novo protein synthesis of the 24 K protease is not apparent in mammalian cells exposed to CPMV. d RAW 264.7 macrophages were transfected with CPMV RNA; both CPMV coat proteins and 24 K protease cannot be detected. Positive controls: CPMV for coat protein detection and CPMV infected plant sap for 24 K protease detection. e Cell lysates from RAW 264.7 macrophages transfected with CPMV RNA or a GFP expression cassette (positive control) were also analyzed by MudPIT analysis: CPMV-related proteins cannot be identified; GFP protein is detected among mouse proteins.