FIG. 4.

Vpx interacts with both importin alpha and importin beta in vivo as well as in vitro. (A) Vpx expression plasmids alone or cotransfected with importin alpha or importin beta expression plasmids in Cos-7 cells. After 16 h of transfection, cell lysates were subjected to immunoprecipitation with monoclonal anti-Vpx antibody, followed by Western blotting with anti-importin alpha (upper panel) or anti-importin beta (lower panel) antibodies. Lane 1, untransfected cell lysates; lane 2, cell lysates from Vpx and importin alpha- or importin beta-cotransfected cells; lane 3, Vpx-transfected cell lysates; lane 4, endogenous importin alpha or importin beta detected by Western blotting with respective antibodies without being subjected to immunoprecipitation. (B) [³⁵S]methionine-labeled Vpx1-40, Vpx1-20, and Vpx65-75 were incubated with GST, GST-importin , and GST-importin beta, which had been immobilized on glutathione-Sepharose. The ³⁵S-labeled HIV-1 Rev NLS, SV40 NLS, alpha, and GFP were used as controls. (C) The input (20%) of the indicated [³⁵S]methionine-labeled proteins was resolved by SDS-15% PAGE and analyzed by autoradiography. (D) Importin alpha and importin beta were expressed and purified as GST fusion proteins as described elsewhere (22). Purified fusion proteins were dialyzed and rebound with glutathione-Sepharose beads, and equal amounts of GST, GST-importin alpha, and GST-importin beta were used in all the in vitro pull-down assays, as indicated by Coomassie blue staining. All the purified fusion proteins were resolved by SDS-10% PAGE. Molecular weights (in thousands) are noted at the left. Imp, importin.