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. 2017 Apr 26;7:1175. doi: 10.1038/s41598-017-01344-9

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© The Author(s) 2017

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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p21 is a target of CRL4B^DCAF11 E3 ligase in human osteosarcoma cells. (A) Flag-tagged p21 associated with DCAF11 in vivo. The pCDNA3-p21-Flag plasmid was transformed into hFOB1.19 (1), U2OS (2) or Saos-2 (3) cells. After 48 h incubation, Flag-tagged proteins were immunoprecipitated with either protein A agarose or anti-Flag-agarose. The cell lysate and pull-down products were analysed by immunoblotting with anti-p21, anti-DCAF1, anti-DCAF4, anti-DCAF11 or β-Actin antibodies. (B) p21 interacted with DCAF11 in yeast. The pGBKT7-p21 plasmid was co-transformed with pGADT7-DCAF11 or pGADT7-DCAF4 into the yeast strain AH109. Growth of the transformed yeast was assayed on media lacking Trp and Leu (top panel) or lacking Trp, Leu, and His (bottom panel). Columns in each panel represent serial 10-fold dilutions. (C) p21 was significantly down-regulated in human osteosarcoma cell lines. Cell lysates were applied to WB analyses with specific antibodies against p21, CUL4B, p27, or β-Actin. (D) p21 was ubiquitinated by CRL4B^DCAF11 E3 ligase in vitro. The purified His-p21 protein was incubated with E1, with or without E2, and with or without CUL4B-Flag in reaction buffer, followed by immunoblotting for p21.