Fig. 4.
Trn interacts with CiN and is required for NLS-N-mediated Ci nuclear localization. (A) Schematics of human Kapβ2, Trn and Drosophila protein encoded by CG8219 with various domains demarcated by colored boxes. Note the overlap between ARM and Heat-like repeats. Lines underneath each protein indicate the regions targeted by the corresponding RNAi constructs. (B) Western blot analysis to confirm the targeting efficiency and specificity for the indicated RNAi constructs. (C) Knockdown of Trn but not CG8219 affects CiN nuclear localization. S2 cells pre-treated without or with the indicated dsRNA were transfected with Myc–CiN expression construct and immunostained with an anti-Myc antibody and Phalloidin. For each treatment, 100 cells were randomly picked and categorized based on the differential nuclear and cytoplasmic distribution of Myc signal. (D) Knockdown of Trn affects nuclear localization of full-length Ci in a manner dependent on the intact NLS-N. S2 cells pre-treated with control or TrnC dsRNA were transfected with HA–Ci, HA–CimNLS-C or HA–CimNLS-N. After treatment with LMB, transfected cells were immunostained to visualize subcellular localization of Ci. (E–G″) Late third instar wild-type wing disc (E,E″) or wing discs expressing UAS-Smo-PKA12 either alone (F,F″) or together with UAS-Trn-RNAi (G,G″) with MS1096 were immunostained to show the expression of Ci (red) and Ptc (blue). Trn RNAi enhanced the ptc expression defect caused by overexpression of Smo-PKA12. (H–I′) Low and high magnification (inset) views of a wild-type wing disc (H) and a wing disc expressing HA–Trn with MS1096 (I,I′) treated with 50 ng/ml LMB for 2 hours prior to immunostaining with anti-Ci (red) and anti-HA (green) antibodies. Arrows indicate the anterior compartments. In wild-type disc, LMB treatment leads to Ci nuclear accumulation only in anterior cells abutting the A/P boundary (H). Overexpression of Trn promotes Ci nuclear localization in A-compartment cells distant from the A–P boundary (I,I′). (J) Trn but not CG8219 binds CiN in a manner dependent on the PY-NLS. Purified Myc-tagged CiN or its variants were incubated with immunopurified HA–Trn or HA–CG8219. The protein complexes were immunoprecipitated with an anti-Myc antibody, followed by western blot analysis.