Figure 3

NLS mutant ARX co-immunoprecipitates and co-localizes with N-terminally truncated IPO13 in mammalian cells. (A) HEK293T cells transfected with Myc-ARX-Wt or NLS mutant constructs and V5-IPO13 were lysed and protein immunoprecipitated (IP) with antibodies against the V5 or Myc tags. Samples were loaded onto 4%-12% SDS-PAGE gels and analysed for the presence of co-IP proteins. Reciprocal Co-IPs was conducted on replicate samples; IP with rabbit anti-V5 antibody (top panel) and detection of Myc-ARX proteins bound to V5-IPO13 by immunoblotting with mouse anti-Myc HRP conjugated antibody. All samples transfected with V5-IPO13 showed a protein band of the correct size upon blotting with anti-V5 horseradish-peroxidase (HRP) conjugated antibody. IP with mouse anti-Myc antibody (middle panel) and detection of V5-IPO13 protein bound to Myc-ARX by immunblotting with mouse anti-V5 HRP conjugated antibody. All samples transfected with Myc-ARX showed a protein band of the correct size upon blotting with anti-Myc HRP conjugated antibody. Specific IP of each over-expressed protein was achieved with no band present in samples from cells transfected with both Myc-ARX and V5-IPO13 but no IP antibody added (*). Cells transfected with Myc-ARX alone or V5-IPO13 alone was used as negative controls. V5-IPO13 (~88.6 kDa) and Myc-ARX (~62.2 kDa) are both present in protein lysates (bottom panel). (B) Pictomicrographs showing V5-IPO13 co-localises with ARX-Wt and mutant ARX protein (indicated on left of panels) in both nuclear and cytoplasmic aggregates in cells co-transfected with Myc-ARX and V5-IPO13 constructs for 24 h. ARX detected by anti-ARX antibody and FITC conjugated secondary antibody (left panel), V5-IPO13 detected by anti-IPO13 antibody and Cy3 conjugated secondary antibody (middle panel) and merged image including blue DAPI stained nuclear material (right panel). Scale bar = 10 μM.