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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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CRL4B^DCAF11 E3 ligase ubiquitinated p21 at K16, K154, K161 and K163. (A) Possible ubiquitination sites of p21. The amino acid sequence of p21 contains six lysines. (B,C) CRL4B^DCAF11 E3 ligase ubiquitinated p21 at K16, K154, K161 and K163 in vitro. Purified His-p21 and p21 mutants, including p21^K16R, p21^K75R, p21^K141R, p21^K154R, p21^K161R, p21^K163R, p21^{K154R K161R K163R}, p21^{K16R K161R K163R}, p21^{K16R K154R K163R}, p21^{K16R K154R K161R} and p21^{K16R K154R K161R K163R}, were incubated with E1, E2, and CUL4B-Flag in ubiquitination reaction buffer, followed by immunoblotting for p21. (D) CRL4B^DCAF11 E3 ligase ubiquitinated p21 in vivo. U2OS cells were transfected with p21-Flag and HA-Ubiquitin, followed by retroviral infection of CUL4B shRNA. After 48 h, cells were lysed, immunoprecipitated with Flag antibody, and then probed with anti-HA antibody for ubiquitinated p21. Right, demonstrated knockdown of CUL4B by shRNA. (E) CRL4B^DCAF11 E3 ligase ubiquitinated p21 at K16, K154, K161 and K163 in vivo. U2OS cells were transfected with HA-Ubiquitin and p21-Flag or mutations, including p21^K75R-Flag, p21^K141R-Flag, p21^{K154R K161R K163R}-Flag (K16), p21^{K16R K161R K163R}-Flag (K154), p21^{K16R K154R K163R}-Flag (K161), p21^{K16R K154R K161R}-Flag (K163) and p21^{K16R K154R K161R K163R}-Flag (4 M). After 48 h, cells were lysed, immunoprecipitated with Flag antibody, and then probed with anti-HA antibody for ubiquitinated p21.