Figure 2

Efficiency of F2A cleavage in GFP-F2A-CherryFP context. F2A sequences of various lengths were used to co-express GFP and CherryFP proteins from pGFP-F2A-CherryFP constructs (a, schematic presentation) in vitro using TnT coupled transcription/ translation rabbit reticulocyte lysates (b) and transfected HeLa cells (c). For TnT, reticulocyte lysates were programmed with 20 ng of plasmid DNA and translation products were resolved by the 12% SDS-PAGE. For in vivo studies, HeLa cells were transfected with 1.5 μg of plasmid DNA and harvested 30 h post transfection. Cells were lysed in RIPA buffer and equal amounts of total protein for each transfection were loaded onto 12% SDS-PAGE gel. The proteins were transferred onto a nitrocellulose membrane, blocked in PBST containing 5% milk and probed with anti-GFP (upper blot) and anti-CherryFP (lower blot) antibodies overnight at 4°C. Detection of bound primary antibody was achieved by using respective secondary antibodies, followed by ECL detection. (d) Expression of fluorescent proteins was detected in transfected HeLa cells at 24 h and 48 h post transfection in Evos microscope using 4 × objective. (e) Cellular co-localisation of [GFP-F2A] and CherryFP 30 h post-transfection. HeLa cells pre-plated on cover slips were transfected with 0.5 μg of plasmid DNA and incubated for 30 h, fixed with 100% methanol and mounted using VECTASHIELD mounting medium with DAPI. The images were acquired with Deltavision microscope using 100 × objective using Resolve 3D software. All experiments were done in triplicates.