Figure 4.

Interaction of NS and NPM by both coimmunoprecipitation and yeast two-hybrid analysis. (A) Coimmunoprecipitation of NS and NPM. Proteins captured from U2OS cell extracts by NPM antibody (middle lane) or nonimmune IgG (left lane) were subjected to immunoblotting for NS or NPM. Right lane, immunoblot of total cell protein. The two regions of the blot containing the NS and NPM antibody-reactive bands are juxtaposed in this composite figure. (B) Yeast two-hybrid analysis of NS-NPM interaction. Shown are the β-galactosidase signals for strains carrying pBD-NPM and pAD (activation domain) vector alone (top left), pBD-NPM and pAD-NS (top right), and pBD-NPM and pAD-NPM (bottom left). (C) Mutants of NS. “Basic” denotes the N-terminal domain previously implicated in nucleolar localization, and “G1” and “G4” indicate GTP binding domains. The + and − signs at the right indicate the interaction of each mutant with NPM, as determined by two-hybrid analysis (D). (D) Yeast two-hybrid analysis of NPM interaction with mutant forms of NS.