Fig. 2

Posttranslational modification of Tax by urmylation regulates the nuclear-cytoplasmic shuttling of Tax, and in extension its ability to activate NF-κB signaling. a Co-expression of Flag-Urm1 (red) and Myc-Tax (green) in 3rd instar larval Drosophila salivary glands (employing Sgs-GAL4 as driver) results in a complete blockage of the nuclear Myc-Tax accumulation, which is observed upon expression of Myc-Tax alone. Quantification is shown in (b), n = 12, P < 0.0001. c Verification of the expression levels of Myc-Tax and Flag-Urm1, induced by the UAS/GAL4 system in Drosophila, by Western Blot. d RNAi-mediated knockdown of Urm1 promotes a significant increase in the amount of nuclear Tax, analyzed in the 3rd instar larval wing disc of flies with the indicated genotypes. Engrailed-GAL4 was used to drive expression of Myc-Tax (green) alone (top) and together with Urm1-RNAi (red) (bottom) specifically in the posterior half of the wing disc (left side of each image), while preserving wild-type tissue in the anterior part (right side of each image). The images are taken at the border between the anterior and posterior side, thus displaying the control wild-type tissue as well as the genetically modified area expressing Myc-Tax and/or Urm1-RNAi in the same view. Quantification of Tax/DAPI colocalization is shown in (e), n = 14, P < 0.0001. f Verification of the GAL4/UAS-mediated induction of Myc-Tax expression and the efficiency of RNAi-mediated knockdown of Urm1 in Drosophila, visualized by Western Blot. g The ability of Tax to induce activation of the NF-κB pathway is strongly correlated with the expression levels of Urm1, as indicated by qRT-PCR analysis of Diptericin, an established transcriptional target of NF-κB in the adult Drosophila fat body. Expression of Myc-Tax, Urm1-RNAi and Flag-Urm1 was induced by the UAS/GAL4 system, utilizing FB-GAL4 as driver, and the qRT-PCR was performed in triplicates on two biological replicates (P < 0.001). Images depicting Drosophila salivary glands in (a) and wing imaginal discs in (d) were acquired using a Nikon C1 confocal microscope, magnifications, ×60 Plan Apo VC NA 1,40 oil and ×100 Plan Apo VC NA 1,40 oil and EZ-C1 software. The Duolink^® images were acquired as described in Fig. 1