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. 2023 Oct 12;14(10):674. doi: 10.1038/s41419-023-06213-0

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© The Author(s) 2023

Open Access This 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 license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license 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 license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 4 — Two U-2 OS-derived cell lines were mixed. One of them stably expressed both p63 and p73 mutants that exclusively form the p63₂/p73₂ hetero-tetramer (E363K in the p73 OD and K377E in the p63 OD). The other cell line expressed wild type p63 and a p73 mutant that allow only the formation of p73-homo-tetramers (E363R, K370E, E373R, and R390D). Cells expressing the p63₂/p73₂ hetero-tetramer can be identified by an anti-myc antibody (MYC-tag both on p63 and p73), cells expressing only the homo-tetramer forms by an anti-FLAG antibody (FLAG-tag on both p63 and p73). DARPins were visualized via an anti-HA-tag antibody. As can be seen, only cells positive for the anti-MYC antibody were also positive for the anti-HA-tag antibody staining, indicating specific binding by the DARPin with virtually no background staining. The last column shows a merged image of the staining results with the MYC-, FLAG- and HA-tag antibodies. A control DARPin (cDP) showed no staining. DAPI staining is provided in the first column. The upper row provides an image of the same samples shown directly below just with a lower magnification to display a larger area.