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. 2023 Jun 23;14:3762. doi: 10.1038/s41467-023-39374-9

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© This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply 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. 6 — a A model for the assembly of the RBX1-CUL4-DDB1-DCAF ubiquitin complex. N8 NEDD8, U ubiquitin. b Scheme of 6×His-TOP1 pull-down in HCT116 cells for LC-MS/MS. Three DCAF member proteins were enriched in the 6×His-TOP1 overexpressing sample. PSM peptide spectrum match. c HCT116 cells were transfected with indicated plasmids and control siRNA (siControl) or DCAF13 siRNA, followed by CPT treatment (20 μM, 30 min) for DUST assay immunodetection of ubiquitylated TOP1-DPC and total TOP1-DPC using anti-ubiquitin and anti-TOP1 antibodies. d Immunofluorescence in IdU-labeled DCAF13-FLAG overexpressing HCT116 cells. Cells were pre-extraction and DCAF13 foci and IdU foci were monitored by instant structured illumination microscope (iSIM) using anti-FLAG and anti-BrdU antibodies. The scale bar represents 10 μm. Biological independent experiments were repeated two times. e 6×His-tagged TOP1-expressing HCT116 cells were transfected with DCAF13-FLAG overexpression plasmid or empty vector, followed by treatments with DMSO or CPT (20 µM) ± APH (10 μM, pre-treatment for 2 h) for 30 min. His-pull-down samples and cell lysates (input) were subjected to immunoblotting (IB) with indicated antibodies. f Top panel: HCT116 cells were transfected with the indicated plasmids before DMSO or CPT treatment (20 μM, 30 min), followed by proximity ligation assay (PLA) using anti-His antibody and anti-FLAG antibody. The scale bar represents 15 μm. Bottom panel: quantitation of foci per cells of each treatment group as shown in the upper panel. Data are presented as mean ± SD, n = 222 total cells. The p value was calculated using two-tailed Student’s t test. Experiments were repeated three times. g DCAF13-FLAG expressing HCT116 cells were pulse-labeled with EdU for 10 min in the absence or presence of CPT (1 μM), then chased with thymidine for 40 min, followed by iPOND analysis. iPOND pull-down samples and cellular lysates (IP) were subjected to IB with indicated antibodies. In no-click samples, desthiobiotin-TEG azide was replaced by DMSO.