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. 2020 Dec 22;49(2):902–915. doi: 10.1093/nar/gkaa1224

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© The Author(s) 2020. 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 (http://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 5. — Monoubiquitylation promotes SPRTN degradation and autocleavage. (A) Stability of endogenous SPRTN was determined with a cycloheximide-chase experiment in HeLa-T-REx Flp-In cells. Cells were incubated with cycloheximide for the indicated amount of time (with or without a 2-h pre-treatment with the proteasome inhibitor MG132) prior to cell lysis and analysis by western blotting. (B) Polyubiquitylation of stably expressed doxycycline-inducible YFP-SPRTN-Strep or of YFP-SPRTN-UBZ*-Strep was determined in HeLa-T-REx Flp-In cells upon treatment with proteasome inhibitor MG132 for the indicated amount of time prior to cell lysis and analysis by western blotting. (C) Stability of stably expressed doxycycline-inducible YFP-SPRTN-Strep or a linear SPRTN-Ubiquitin fusion (YFP-SPRTN-Ub^LF) was determined in HeLa-T-REx Flp-In cells using a cycloheximide-chase experiment. Cells were incubated in the presence of cycloheximide for the indicated amount of time (with or without a 2-h pre-treatment with the proteasome inhibitor MG132) prior to cell lysis and analysis by western blotting. (D) Indicated YFP-SPRTN-Strep or linear SPRTN-Ubiquitin fusion (YFP-SPRTN-Ub^LF) variants were transiently transfected in HeLa-T-REx Flp-In cells. SPRTN autocleavage fragments were enriched on GFP-trap resins, followed by western blotting against the N-terminal YFP-tag. Western blotting of cell lysates against GAPDH serves as loading control. Asterisks indicate autocleavage fragments. (E) Indicated YFP-SPRTN-Strep variants were transiently transfected in HeLa-T-REx Flp-In cells in combination with Flag-tagged full-length USP7 (WT or the catalytically inactive CS variant) or the empty vector. SPRTN autocleavage fragments were enriched on GFP-trap resins, followed by western blotting against the N-terminal YFP-tag. Western blotting against GAPDH of cell lysates serves as loading control. Asterisks indicate autocleavage fragments. (F) HAP1 cells were treated with increasing amounts of formaldehyde (FA, 0.25, 0.5, 1 and 2 mM) for 2 h (either with or without a 2-h pre-treatment with ubiquitin-activating enzyme E1 inhibitor as indicated) prior to cell lysis and analysis by western blotting. Asterisks indicate autocleavage fragments. (G) HeLa-T-REx Flp-In cells were treated with proteasome inhibitor MG132 for the indicated amount of time prior to cell lysis and analysis by western blotting. Asterisks indicate autocleavage fragments.