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. 2023 Oct 18;51(20):11178–11196. doi: 10.1093/nar/gkad796

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© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 1. — VHL mediates ubiquitin-dependent degradation of SETDB1. (A) Schematic workflow of MS analysis. Whole cell lysates from RCC4 cells stably expressing Flag- tagged VHL (Flag-VHL) and control (Con) were immunoprecipitated using anti-Flag antibodies. Immunoprecipitated proteins were analyzed using mass spectrometry. (B) MS analysis of VHL-associated proteins. (C) PLA using either anti-SETDB1/anti-VHL antibodies or anti-IgG/anti-VHL to determine the association of VHL and SETDB1 in HeLa cells. Representative images of PLA (red) and those merged with DAPI staining (blue) are shown. Scale bars denote 20 μm. (D) Examination of the physical association of endogenous SETDB1 with VHL via immunoprecipitation (IP) against SETDB1 or IgG followed by immunoblotting with the indicated antibodies. (E) Cellular interactions between ectopically expressed SETDB1 tagged with histidine and streptavidin-binding protein (SBP) and CRL2^VHL in HEK293T analyzed via immunoprecipitation (IP) against the SBP epitope followed by immunoblotting with the indicated antibodies. (F) Immunoblot analysis with antibodies against SETDB1 and VHL to determine the effects of VHL KDwith siRNAs (siVHL) in HeLa cells treated with bortezomib (1 μM) for 12 h. (G) Effect of Flag-tagged VHL overexpression (Flag-VHL) on abundance of endogenous SETDB1 and HIF1α in 786O and RCC4 cells assessed by immunoblotting with the indicated antibodies. (H) Downregulation of endogenous SETDB1 ubiquitination by siRNA-mediated KDof VHL (siVHL) in HeLa cells treated with bortezomib (1 μM) for 12 h. Non-targeting siRNA was used as a negative control.