Figure 11.

Scyl1, Xpo-t containing endogenous E. coli tRNA and RanGTP form a complex. GST-Xpo-t (20 μg; 158 pmol) bound to GT-Sepharose was incubated in the presence (lanes 4 and 5) or absence (lanes 6 and 7) of an excess of exogenous yeast mature tRNA (6 μM) and Ran (4 μg; 158 pmol) in the GDP (lanes 4 and 6) or GppNHp (lanes 5 and 7) bound form. The resins were washed and incubated with Scyl1 (14 μg; 158 pmol). The same amount of Ran and Scyl1 was incubated with bound GST (lane 8). The resins were washed and boiled in sample buffer to elute bound proteins. GST-Xpo-t (lane 1), Ran (lane 2), Scyl1 (lane 3) and the eluates were subjected to SDS-PAGE, followed by Western blot analyses to detect GST-Xpo-t (first row), Ran (second row), and Scyl1 (third row). The proteins also were detected directly by SYPRO Ruby staining of the blot (fourth row).