Fig 7.

BGLF4 interacts with FG repeat-containing nucleoporins. (A and B) HeLa cells were transfected with plasmids expressing BGLF4, d(386-393), d(378-389), d(410-419), or BGLF4 (5A). At 24 h posttransfection, cells were harvested and lysed in RIPA buffer. Protein complexes were immunoprecipitated with BGLF4-specific antibody 2224 (A) or mAb414 (B) and the immunocomplexes were detected in immunoblots using BGLF4-specific antibody 2224 and mAb414 antibody. (C) Purified GST-BGLF4, GST-K102I, GST–importin-β, or GST control was incubated with [³⁵S]Met-labeled Nup62 and Nup153. Proteins that interacted with various GST fusion proteins were analyzed by SDS-PAGE and detected by autoradiography. (D) HeLa cells were permeabilized with digitonin and incubated with purified yeast recombinant GST-LacZ-NLS, GST-BGLF4, or GST-K102I in the absence (left) or presence (middle) of cytosolic factors; (right) permeabilized cells were incubated with 250 μg/ml WGA for 15 min before adding the nuclear import substrates. After 1 h of incubation, cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% TNX-100. GST-BGLF4 and GST-K102I were detected using BGLF4-specific antibody 2224, and GST-LacZ-NLS was detected with anti-GST antibodies.