Testing 2A constructs in HeLa cells. (A) Constructs Tom-2A-GFP and Tom-m2A-GFP used to test the function of the 2A linker. A membrane localised red fluorescent protein (Myr-TdTomato) linked to a nuclear localised green fluorescent protein (H2B-GFP) by either a functional 2A linker (top), or by a non-functional m2A linker (bottom). The specific nucleotide and amino acid changes made in Tom-m2A-GFP are shown in red. (B-B") HeLa cells lipofected with Tom-2A-GFP. Fluorescent cells show segregation of the TdTomato and enhanced green fluorescent protein (EGFP) to mutually exclusive cellular compartments. (C-C") HeLa cells lipofected with Tom-m2A-GFP as a negative control. The EGFP and TdTomato are no longer segregated properly, consistent with them remaining linked together as a single fusion protein due to the mutations in the 2A linker. (B, C) EGFP fluorescence. (B', C') TdTomato fluorescence. (B", C") Merge of EGFP (green) and TdTomato (magenta) fluorescence. Diagram in (A) not to scale. The scale bar in (B) represents 50 μm and applies to panels B through C".