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. 2017 Jan 18;12(1):e0169988. doi: 10.1371/journal.pone.0169988

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© 2017 Bonacci et al

This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Fig 4 — (A) Workflow of the protocol used to identify NUB1 ubiquitinated lysine. (B) METTRE SPECTRES (C) HEK-293T cells were transfected with wild type (WT), K134R, K159R or K134,159R (2KR) mutants Myc-NUB1 together with 6HF-Ubiquitin and Mdm2 constructs. 6HF-Ubiquitinated proteins were isolated by Ni2+ pull down and NUB1 was revealed by western blotting using an anti-NUB1 antibody. (D) Lysates from HEK-293T cells expressing Mdm2 alone or together with wild type (WT), K134R or K159R mutants Myc-NUB1 were subjected to immunoprecipitation with the anti-Myc 9E10 antibody. Immunoprecipitates and input samples were analyzed by western blotting using an anti-Mdm2 antibody.