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. 2018 Mar 29;9:1285. doi: 10.1038/s41467-018-03588-z

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© The Author(s) 2018

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. 3 — USP52 opposes ASF1A polyubiquitination. a In vitro deubiquitination assays with HeLa cells-purified Myc-USP52 (1, 2, and 3 μg for each lane) and HA-Ub-conjugated FLAG-ASF1A with high salt and detergent buffer. b HeLa cells stably expressing FLAG-ASF1A were co-transfected with control siRNA or USP52 siRNA together with HA-Ub as indicated. Cellular extracts were prepared for co-immunoprecipitation assays with anti-FLAG followed by IB with anti-HA. c HeLa cells stably expressing FLAG-ASF1A were co-transfected with HA-Ub and different amounts of Myc-USP52. Cellular extracts were prepared for co-immunoprecipitation assays with anti-FLAG followed by IB with anti-HA. d HeLa cells were transfected with different amounts of Myc-USP52 and cellular extracts were prepared for co-immunoprecipitation assays with anti-ASF1A followed by IB with antibody against ubiquitin. e Cellular extracts from HeLa cells expressing HA-Ub/K48-only and different amounts of Myc-USP52 were prepared for co-immunoprecipitation assays with anti-ASF1A followed by IB with anti-HA. f In vitro deubiquitination assays with HeLa cells-purified FLAG-tagged ASF1A-Ub/K48-only and different amounts of Myc-USP52 (1, 2, and 3 μg for each lane) with high salt and detergent buffer. g HeLa cells stably expressing wild-type ASF1A (ASF1A/wt) or different K to R mutants were co-transfected with HA-Ub and control vector or Myc-USP52 as indicated. Cellular extracts were prepared for co-immunoprecipitation assays with anti-FLAG followed by IB with anti-HA. h Mass spectrometry analysis of ASF1A ubiquitin conjugation sites. HeLa cells stably expressing FLAG-ASF1A were co-transfected with HA-Ub and USP52 siRNA. Cellular extracts were collected and sequentially purified with anti-FLAG affinity gel and HA affinity gel to enrich HA-Ub-conjugated ASF1A. After trypsinization, the retrieved peptides were subjected to mass spectrometry analysis. Fragmentation spectrums and parameters of the identified ASF1A peptides with di-Glycine remnant are shown. Detailed results from the mass spectrometric analysis are provided as Supplementary Data 2