FIGURE 8.

GST pulldown assay of karyopherins α3 and α4. A, bars represent the constructs used. B, shown is a Coomassie gel after purification of GST (gray arrowhead) and GST fused to the TM3–4 loop of human GlyRα1 (GST-α1, black arrowhead). C, shown is a Western blot with anti-T7-antibody after bacterial expression of karyopherin α3 (K3) und α4 (K4) with a T7 tag. Full-length (F, black arrow) and N-terminal-truncated (T, gray arrow) constructs lacking the autoinhibitory domain are shown. D, Western blot with an anti-T7-antibody detecting full-length and truncated karyopherins pulled down with GST and GST-α1. Both karyopherins specifically bind to the TM3–4 loop in vitro. Binding is not hindered by the presence of the autoinhibitory domain.