scFvs specifically bind glypican-3-expressing cell lines. A. Western blot confirmation of expression of glypican-3 in HepG2 and 293T.GPC3, knockdown in HepG2.sh57, and absence of expression in parental 293T and Hs578t. 1G12 antibody used for detection. B. Detection of hGPC3 expression on HepG2 and Hs578T cells by immunofluorescent microscopy using commercial 1G12 anti-GPC3 mAb followed by anti-mouse Alexa Fluor 488. C. Binding of scFvs to HepG2 and Hs578T cells by flow cytometry using indicated scFv or 1G12 mAb followed by either APC-conjugated anti-V5 mAb or anti-mouse IR680 (for 1G12 only). Cells incubated with isotype antibody were used as negative control (grey area). D. Immunofluorescence of scFvs (detected with anti-V5 Alexa Fluor 488 (green)) detected on HepG2.tdTomato (red).