FIGURE 5.

Nuclear CTSV trafficking is mediated via a bipartite nuclear localisation signal. (A) In silico analysis via NLS mapper has identified a potential bipartite NLS sequence motif (underlined residues) within CTSV. A CTSV expression construct with a mutated NLS was generated (mtNLS) by mutation of the basic lysine and arginine residues within the NLS motif to alanines (indicated in red). (B) Western blotting analysis following transfection of the mtNLS expression construct shows reduced nuclear CTSV protein expression in MCF-7 and ZR75-1 cells. (C) Confocal analysis of CTSV protein expression (red) following transfection of the mtNLS construct was used to confirm reduced nuclear CTSV expression. DAPI counterstaining (blue) was used to identify cell nuclei. Scale bars = 10 μm (D) MCF-7 and ZR75-1 cells were treated with ivermectin to confirm the functionality of the predicted NLS motif. Western blotting analysis of cytoplasmic (C) and nuclear (N) fractions confirmed nuclear localisation of CTSV was reduced following ivermectin treatment. HDAC1 and tubulin expression were included as loading controls relative to the nuclear and cytoplasmic fractions respectively and data presented is representative of three independent experiments.