Figure 4.

Efficiency of F2A cleavage in pGFP-F2A-CherryFP “mutants” with -SGSRGAC- substitution immediately upstream of F2A of 25–18aa. pGFP-F2A-CherryFP “mutant” constructs were used to coexpress GFP and CherryFP proteins from a single ORF in transfected HeLa cells. The cells were transfected with 1.5 μg of plasmid DNA and harvested 30 h after 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 PBS containing 5% milk, and probed with anti-GFP (upper blot) and anti-CherryFP (middle blot) antibodies overnight at 4°C. Detection of bound primary antibody was achieved by using respective secondary antibodies, followed by ECL detection. All experiments were done in triplicate.