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. 2021 Dec 13;50(11):6001–6019. doi: 10.1093/nar/gkab1189

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© The Author(s) 2021. 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-NonCommercial License (https://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

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Figure 2. — Tissue-specific expression of the Tj-TR isoform in Drosophila. (A) Sequence map of chromosomal modifications introduced in the tj locus by CRISPR/Cas9 genome editing. (B–E) Adult brains and ovaries of tj-TR mutants; each column depicts brains (upper panels) and germaria (lower panels) of the indicated genotypes. For both organs, Tj and Flag are shown in single channels below the merged images. (B) Control tissues express no Flag. Endogenous Tj is expressed in the brain, especially in three distinct clusters (arrows) and in several types of somatic cells in the germarium. (C) tj^nat/nat brains express Tj in a pattern indistinguishable from control. In addition, brains express the Flag-tagged TR isoform, demonstrating the occurrence of TR in neural cells. In germaria, Tj appears as in control, but no Flag staining is detectable. (D) Overlapping pattern of Tj and Flag staining in tj^TR/TR brains and germaria, consistent with constitutive induction of TR in Tj. (E) Abrogation of TR in Tj observed in tj^nTR/nTR brains and germaria, evidenced by the lack of Flag stain. Scale bars: 100 μm for brains; 10 μm for germaria. (F) Western blot with anti-Flag antibodies showing the relative abundance of Flag-tagged Tj-TR isoform in adult tissues from tj^nat/nat and tj^TR/TR mutants. (G) TR efficiency estimated in adult heads and ovaries using quantitative western blot using total protein normalization. P-values were calculated using unpaired Student's t-test obtained from three biological replicates (*P< 0.05, **P< 0.005, ***P< 0.0005). (H) RT-qPCR analysis of tj transcripts using cDNA prepared from adult heads of tj^nat/nat, tj^TR/TR and tj^nTR/nTR mutants. Error bars represent the upper and lower limit of RQ defined by the standard deviation of ΔΔC_T from three biological replicates. The data were normalized against average ΔC_T of the housekeeping gene αTub84B. Two-tailed unpaired Student's t-tests were performed from the ΔΔCT values of tj^nat/nat vs tj^TR/TR and tj^nat/nat versus tj^nTR/nTR (n.s., not significantly different from control).