Fig. 5.
Exportin pulldown by LE-GST. LE-GST proteins (or GST alone) were incubated with HeLa cell cytosol then reacted with glutathione-conjugated beads in assays identical to those previously described (24). The extracted proteins were fractionated by SDS/PAGE and then identified by Western analyses. The antibodies included αCrm1 (Abcam 24189; 1:2,000) αCAS (sc-1708, 1:500; Santa Cruz Biotechnology), and αGST (GE Healthcare; 1:10,000). LE-GST proteins made this way (16) can show additional bands from alternative translational start sites. Similar pull-down assays were used in the original identification of L:Ran interactions (24), albeit protein detection was with different antibodies.