Figure 7.

Arx1 interacts with nucleoporins. (A) A panel of plasmids that express Arx1 or the Nup116 FG domain, as a control, fused to the Gal4-binding domain (BD) and various Nup FG domains fused to the Gal4 activation domain (AD) were transformed into yeast strain BJ69-4A and spotted onto selective media (Leu⁻ Ura⁻ His⁻ dropout for Arx1-Nup interactions or Leu⁻ Trp⁻ His⁻ dropout for Nup-Nup interactions) to test for potential interactions. Plates were incubated at 30°C for 4 d. Positive interactions should drive expression of the HIS3 reporter. The indicated concentrations of 3-AT were added to increase the stringency of the assay. (B) In vitro interaction of Arx1 with GST-Nup fusions. GST, GST-Nup100(aa 1–640), GST-Nup116(aa 165–716) and GST-Nsp1(aa 1–603)-coated beads (2 μg GST fusion protein in 10 μl of beads) were incubated with purified Arx1 (0.8 μg) in the absence or presence of RNase A (25 μg). After 1 h at 4°C, beads were concentrated by centrifugation, washed twice, and bound proteins eluted first with 0.3 M MgCl₂, followed by elution with SDS. Bound proteins were resolved by SDS-PAGE and visualized with Coomassie blue. The bands present below the full-length GST-Nup fusion proteins in the lanes without Arx1 are degradation products of the fusion proteins.