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. 2021 Mar 25;21:321. doi: 10.1186/s12885-021-08056-4

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© The Author(s) 2021

Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

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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)