Figure 2
Uptake of Phylomer CPP_EBD_S11 fusion proteins validated by GFP complementation. (a) Thirteen Phylomer peptides showed a positive GFP complementation signal, evidence of intracellular delivery. These functionally validated CPPs were of various sizes, net charges and origin. (b,c) Dose-dependent uptake of recombinant Phylomer CPP_EBD_S11 fusion proteins was determined by GFP complementation in (b) HEK-293 or (c) CHO-K1 cells transiently transfected with a hGFP1–10 expressing plasmid. (d,e) Dose-dependent uptake of recombinant CPP fusion proteins was confirmed in stable cell lines by GFP complementation of (d) EBD_S11 proteins in HEK-293/GFP1–10 and (e) TRX_S11 proteins in CHO-K1/GFP1–10 where hGFP1–10 is stably expressed. The greater sensitivity of the stable cell lines enables comparison of uptake at low-dose concentrations. “No CPP” control is shown at the highest concentration (40 µM). In flow cytometry experiments GFP complementation is measured as % fluorescent cells. “No CPP” control is EBD_S11 (b-d) or TRX_S11 (e) protein with no CPP moiety. Results are representative of two independent experiments. Error bars represent standard deviation from the mean between duplicates. (f) Fluorescence microscopy visually confirms the CPP-dependent GFP complementation (FITC channel) in CHO-K1 cells transiently transfected with GFP1–10, comparing 0084_EBD_S11 fusion protein (10 µM) to the “No CPP” _EBD_S11 control protein (10 µM). Cells are counter-stained for endogenous cytoplasmic β-Actin (TRITC) and nuclei (DAPI); bar scale is 25 µm.
