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. 2014 Apr 11;42(9):5765–5775. doi: 10.1093/nar/gku225

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© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 2. — E6/E7 expression enhanced BMAL1 ubiquitination and proteasomal degradation through UBE3A. (A) Left panel: IB of steady state BMAL1 in WT and E6/E7 cells. Right panels: integrated density measurement of BMAL1 (n = 3, P < 0.05) and steady state Bmal1 gene expression by qPCR (n = 3, P > 0.05). **P < 0.01, see underlined. (B) Top panel: BMAL1 protein level following cycloheximide (20 μg/ml). Bottom panel: protein degradation rate. (C) Ubiquitination assay of V5-BMAL1 in HEK 293T cells by nickel affinity pull-down. Total levels of V5-BMAL1 in the input lysates are shown in the panel below. MG132 (5 μM) was applied in all cases. (D) V5-BMAL1 levels in HEK 293T cells, transfected with E6/E7, co-transfected with either HA-UBE3A or a dominant negative mutant (C833A). Representative, n = 3.