Figure 2.

Validation of cargo candidates as true Xpo7 binders. (A) H14-ZZ-bdNEDD8–tagged cargo candidates (human proteins) were immobilized on anti–protein A beads and incubated with a cytoplasmic HeLa extract in the absence or presence of 3 µM RanGTP. After washing off unbound material, the immobilized candidates and bound proteins were eluted by bdNEDP1 protease cleavage. 1/250 of the starting extracts and 1/10 of the eluates were analyzed by SDS-PAGE followed by immunoblotting with an anti-hsXpo7 antibody recognizing a C-terminal epitope (Mingot et al., 2004). (B) Binding assays were performed as in A, but candidates (from mouse) were incubated with purified recombinant Xpo7 in the absence or presence of RanGTP, and the eluates were analyzed by SDS-PAGE and Coomassie staining. Anp32e* corresponds with Anp32e isoform 3 (E9Q5H9). Mw, molecular weight.