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. 2018 Oct 10;46(20):10697–10708. doi: 10.1093/nar/gky835

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© The Author(s) 2018. 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/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 2. — Zeb1 inhibits the in vivo induction of atrogenes upon immobilization. (A) Wild-type and Zeb1 (+/-) mice were subjected to unilateral hindlimb immobilization during 3 and 17 days and their immobilized and non-immobilized gastrocnemius were then examined for Fbxo32 mRNA expression by qRT-PCR with respect to Gapdh. Fbxo32 mRNA levels in the non-immobilized hindlimb at day 0 were arbitrarily set to 100 with all other data genotypes and conditions referred to them. Data represent the mean of at least five mice for each genotype and condition. (B) The gastrocnemius of mice from both genotypes after 3 days of the unilateral hindlimb immobilization protocol were stained with antibodies against Atrogin-1 (clone AP2041) and laminin (clone 48H-2), and counterstained for 4′,6-diamidino-2-phenylindole (DAPI) for nuclear staining. Captures for single immunostaining are shown in Supplementary Figure S2A. Scale bar: 50 μm. (C) As in (A), but for Trim63. (D) As in (B), but the lysates from gastrocnemius of mice from both genotypes after 3 days of the unilateral hindlimb immobilization protocol were blotted for MuRF1 (clone C11) along with GAPDH (clone 14C10) as loading control. See Supplementary Figure S2B for full unedited blots. The blots shown are representative of three independent experiments. (E) Wild-type and Zeb1 (+/-) mice were subjected to the unilateral hindlimb immobilization protocol for 3, 5 and 17 days. At the end of each time point, they were euthanized and mRNA levels for Psma1, Ctsl, Gabarapl1, 4ebp1 and Nrf2 were assessed by qRT-PCR. For each gene, mRNA levels shown correspond to that in the gastrocnemius of the immobilized with respect to the contralateral non-immobilized hindlimb. The gene expression in the non-immobilized gastrocnemius at days 3, 5 and 17 was similar than that at day 0 shown. At least five mice from each genotype and day were analyzed.