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. 2023 Nov 17;14:7473. doi: 10.1038/s41467-023-43038-z

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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 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/.

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Fig. 8 — a Nanobody pull-down and antigen identification pipeline using immunoprecipitation. b Western blot of α-PHB2 protein after pulled-down by Nb-FC fusion protein (IgGFC-Nb1) (representative of 3 independent experiments) c Dot plot for Phb and Phb2 expression across immune cell populations. d Violin plot showing the Phb2 signaling pathway across immune cells populations, revealing the greatest expression in the DC CD103⁺/Xcr1⁺ population (red box). e Representative confocal images from two independent experiments of Nb1 and PHB2 colocalization in MC38 cells with the upper row Nb1/α-PHB2 stained and lower row secondary and tertiary antibody alone control. The first column is the nanobody channel red, then α-PHB2 green, and the overlay with Höchst 33342 blue (scale 5 μm). f Representative confocal images from two independent experiments of Nb1 colocalization with membranous regions expressing PHB2 from 20 different cells (white arrows). g Mander’s colocalization coefficient M1 of Nb1 pixel overlapping PHB2 pixel (mean represented by black line) for Nb1 and α-PHB2 (n = 20 images), α-PHB2 antibody alone (n = 6) and 2nd/3rd antibodies alone (n = 6) from two independent experiments with six incrementing pixel shift configurations. Source data are provided as a Source Data file.