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. 2021 Sep 2;11:17587. doi: 10.1038/s41598-021-97060-6

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© The Author(s) 2021

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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Biotinylation of endogenous proteins in MCF7 cells. MCF7 cells were transiently transfected for 24 h with an expression vector (pcDNA3.1) bearing the cDNA for 3F-CXXC5, 3F-CXXC5-BirA*-HA, or BirA*-HA. Cells were then treated without (-) or with ( +) biotin (50 μM) and ATP (1 mM) for 16 h. (a) Transiently transfected cells were subjected to immunocytochemistry using the Flag, the HA, or the Biotin antibody followed by an Alexa Fluor 488 (green fluorescein) conjugated goat anti-mouse IgG for the Flag antibody, or an Alexa Fluor 594 (red fluorescein) conjugated goat anti-rabbit IgG for the HA and the Biotin antibody. The scale bar is 20 µm. (b) Total protein extracts of MCF7 cells were subjected to SDS-10%PAGE followed by WB using the Flag, the HA, or the Biotin antibody followed by an HRP-conjugated goat-anti mouse secondary antibody for the Flag (Advansta R-05071–500) or goat-anti-rabbit secondary antibody for the HA or the Biotin antibody (Advansta R-05072–500). Molecular masses (MM) in kDa are indicated.