Fig. 3
MPJSBWMV self-interacts. A-D IP was conducted with leaf-material from N. benthamiana co-expressing MPJSBWMV:RFP and one of the GFP-tagged proteins, respectively, using RFP-trap antibodies. Western blots with anti RFP (A) and anti GFP (B) antibodies were conducted with crude protein extracts to demonstrate that all proteins were expressed. A MPJSBWMV:RFP was expected at approximately 70 kDa. The bands at approximately 50 kDa in the input sample likely represent unspecific antibody binding. B anti GFP antibodies detected the MPJSBWMV:GFP at approximately 70 kDa, PDLP1:GFP at approximately 65 KDa and GFP at approximately 27 KDa. C Western blots conducted with immunoprecipitated samples and probed with RFP-specific antibodies revealed the presence of MPJSBWMV:RFP (approximately 70 kDa) in all samples. D Western blots conducted with immunoprecipitated samples were probed with anti GFP antibodies, a band for MPJSBWMV:GFP (approximately 70 kDa) was detected while no bands for PDLP1:GFP (approximately 65 kDa), and for GFP (approximately 27 kDa) were visible. E–G Fluorescence lifetime of MPJSBWMV:GFP expressed in N. benthamiana epidermal cells was measured by FRET-FLIM. The images reveal fluorescence lifetime in a pseudo-color scheme, ranging from 1.8 ns (blue) to 2.7 ns (orange) as shown in the color-coded bar at the right hand side. The fluorescence lifetime of (E) MPJSBWMV:GFP expressed alone, (F) MPJSBWMV:GFP in the presence of MPJSBWMV:RFP, (G) MPJSBWMV:GFP in the presence of PDLP1:RFP