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. 2016 Oct 17;213(11):2503–2523. doi: 10.1084/jem.20160397

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© 2016 Delaunay et al.

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Figure 6. — The ITAF DEK is a direct target of U34 tRNA modifications. (A) Table of reported LEF1 ITAFs (Tsai et al., 2011). Proteins were ordered according to the combined frequency of AAA, GAA, and CAA codons in their ORF. (B) Western blot showing indicated proteins expression in control (shCTR) or ELP3-depleted (shELP3#1 and shELP3#2) MDA-MB-231 cells. HSP90 is used as loading control. (C) RT-qPCR assessing DEK mRNA levels in control or ELP3-depleted MDA-MB-231 cells. Values represent mean ± SD of three different experiments performed in triplicate. (D and E) Expression of LEF1 and DEK was assessed at protein levels by Western blot (D and F) and at mRNA levels by RT-qPCR (E and G) in control or ELP3-depleted MCF7-M cells. (F–I) DEK and LEF1 protein (F and H) and mRNA (G and I) expression were assessed in control or CTU-1–depleted MDA-MB-231 (F and G) and MCF7-M cells (H and I; ***, P < 0.001, Student’s t test). (J) Western blot showing DEK protein expression in extracts from Elp3^CTRPyMT and Elp3^ΔMECPyMT primary tumors of similar size. HSP90 is used as loading control. (K) IHC staining for Dek expression in tumorigenic mammary glands from 7-wk-old Elp3^CTRPyMT or Elp3^ΔMECPyMT mice. Images were generated from five different tumors/mouse, using three mice per condition. Representative images are shown. (L) The mean Dek-specific signal shown in M was quantified and plotted (mean values ± SD; Student’s t test; ***, P < 0.001). (M) Positions of AAA, CAA, and GAA codons were represented in the DEK ORF sequence. (N) DEK (FLAG), LEF1, ELP3, and CTU1 protein levels were assessed in control (shCTR), ELP3 (shELP3)-, or CTU1 (shCTU1)-depleted MDA-MB-231 stably expressing DEK-wild-type sequence (DEK-WT-FLAG) or a DEF mutant in which AAA, CAA, and GAA codons have been replaced by their synonymous codons (AGA, CAG, GAG, respectively; DEK-δAAA/GAA/CAA-FLAG).