Fig. 3

RGD4C-p21Ras-scFv immunoreactivity toward p21Ras and the effect on tumor cell penetration. (a) Both ELISA (left) and WB (right) revealed that RGD4C-scFv had almost the same immunoreactivity to p21Ras as the anti-p21Ras scFv and RGD4C-linker-scFv. (b) Immunocytochemistry showed that SW480 cells treated with RGD4C-p21Ras-scFv were positively stained, while the anti-p21Ras scFv and PBS incubation groups were negative. We demonstrated that RGD4C-scFv and RGD4C-linker-scFv entered SW480 cells. (c) The in vitro tumor targeting of the fusion protein was analyzed by WB. SW480, Huh7, U251, and A549 tumor cells with high integrin expression and normal BEAS-2B cells without integrin expression were cocultured with 20 μM RGD4C-scFv or RGD4C-linker-scFv for 5 h. RGD4C-scFv and RGD4C-linker-scFv were detected in the tumor cells but not in BEAS-2B cells. (d) Immunofluorescence detection showed co-localization of the internalized RGD4C-scFv with p21Ras protein in SW480 cells. Red immunofluorescence was observed on the of tumor cells with p21Ras protein, and green immunofluorescence was RGD4C-scFv. Nuclei were counterstained with DAPI (blue)