Endogenous IPO13 co-localizes and co-immunoprecipitates with mutant ARX protein. (A) Pictomicrographs showing sub-cellular localization of Wt and mutant Myc-ARX detected by anti-ARX antibody and FITC conjugated secondary antibody (left panel), endogenous IPO13 detected by anti-IPO13 antibody and Cy3 conjugated secondary antibody (middle panel) and merged image including blue DAPI stained nuclear material (right panel). Endogenous IPO13 co-localises with Myc-ARX mutant protein, including the double mutant R332P-R379L, in both nuclear and cytoplasmic aggregates, but does not co-localize with the ARX-Wt protein (lane 1). (B) HEK293T cells transfected with Myc-ARX-Wt or NLS mutant constructs were lysed and protein IP with antibodies against endogenous IPO13. Samples were loaded onto 4%-12% SDS-PAGE gels and analysed for the presence of co-IP proteins. IP with Goat anti-IPO13 antibody (top panel) and detection of Myc-ARX proteins bound to IPO13 by immunoblotting with mouse anti-Myc HRP conjugated antibody. All samples transfected with Myc-NLS mutant ARX showed a protein band of the correct size upon blotting with anti-Myc HRP conjugated antibody, with no band present in the ARX-Wt lane. Endogenous IPO13 (~108 kDa) and Myc-ARX (~62.2 kDa) are both present in protein lysates (bottom box). Scale bar = 10 μM.