Fig. 3
Nuclear degradation of NICDK2R-HA is reduced. A–B” Expression of NICD-HA and NICDK2R-HA in late third instar wing imaginal discs for 48 h under control of ptc-GAL4 tub-GAL80 ts. A, A’, B, B’ Expression of NICD-HA and NICDK2R-HA induced strong ectopic Wg expression along the entire ptc-Gal4 expression domain in the wing pouch. In contrast to NICD-HA, the Wg expression domain induced by NICDK2R-HA was broader (insert in A’, B’, arrows), indicating that NICDK2R-HA has a stronger ability to activate N target genes, even in regions of low expression of the ptc-Gal4 expression gradient. A”, B” The magnification highlights the nuclear localisation of the NICD variants. C–E Accumulation of NICD-HA and NICDK2R-HA in the nucleus after a pulse of expression of only 10 h using ptc-Gal4 combined with tub-Gal80 ts. E The quantification shows that NICDK2R-HA accumulated more strongly in the nucleus and in more nuclei than NICD-HA (NICD-HA: n = 14 / NICDK2R-HA n = 12 wing imaginal discs, significance test: Student’s t test p ≥ 0.05 (n.s.) / p ≤ 0.05 (*) / p ≤ 0.01 (**) / p ≤ 0.001 (***)). F–H Pulse-chase assay to analyse the degradation of the NICD constructs in the nucleus. To analyse NICD degradation in the nucleus, NICD-HA and NICDK2R-HA expression was pulsed for 10 h and afterwards the larvae were shifted back to a restrictive temperature (18 °C) for 16 h (chase). The remaining HA-positive nuclei in the wing pouch were counted. H The quantification shows that significantly more nuclei remained positive for NICDK2R-HA than for NICD-HA, suggesting that the degradation of NICDK2R-HA in the nucleus was significantly reduced compared to NICD-HA (NICD-HA: n = 17 / NICDK2R-HA: n = 20, significance test: Student’s t test p ≥ 0.05 (n.s.) / p ≤ 0.05 (*) / p ≤ 0.01 (**) / p ≤ 0.001 (***)). It revealed that after the chase a higher percentage of nuclei remain positive in the case of NICDK2R-HA compared to NICD-HA (15.4% vs 12.6%, respectively)