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. 2018 Oct 10;9:4188. doi: 10.1038/s41467-018-06592-5

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© The Author(s) 2018

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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Fig. 3 — Su(z)12 is a target of miR-34 in the brain. a, b Su(z)12 protein levels are deregulated in miR-34 mutants. Su(z)12 protein was detected using a transgene containing the Su(z)12 genomic region tagged with the HA epitope on the C-terminus of the predicted protein (Su(z)12-C-term HA). Su(z)12 levels decreased with age in wild-type, but remained elevated in miR-34 mutants. Protein samples from dissected brains. Su(z)12-C-term HA protein levels were normalized to tubulin loading control. Mean ± SEM, n = 3 biological replicates. Significant main effects were observed (genotype: F_1,8 = 68, p < 0.001 age: F_1,8 = 101, p < 0.001). (*p < 0.05, **p < 0.01, two-way ANOVA with Tukey post-test).c, d The regulation of Su(z)12 protein levels by miR-34 is dependent upon the miR-34 seed sequence in the 3′UTR of the Su(z)12 transcript. The Su(z)12-C-term HA-mutant 3′UTR transgene contains a mutation in the miR-34 seed sequence that relieves the transcript from regulation by miR-34 (see Fig. 1d). Su(z)12-C-term HA-mutant 3′UTR protein levels still decrease with age, but are no longer increased in miR-34 mutants. The difference between wild-type at 20d and miR-34 mutant at 20d is not significant. Protein samples from dissected brains. Quantification of Su(z)12-C-term HA-mutant 3′UTR protein levels using Western immunoblotting, normalized to tubulin loading control. Mean ± SEM, n = 3 biological replicates. Significant main effects were observed (genotype: F_1,8 = 17, p < 0.01 age: F_1,8 = 28.5, p < 0.001). (*p < 0.05, two-way ANOVA with Tukey post-test)