Figure 2

Stau2 interacts directly with Upf1. (A) Recombinant FLAG-Upf1 protein was mixed with either recombinant GST-Stau2 or GST, and the mixtures were incubated at 4 °C for 2 hr. The complexes were precipitated using glutathione-Sepharose beads. Ten percent of the total mixture and 30% of the precipitate were subjected to SDS-PAGE followed by CBB staining. Bands of contaminated Hsp70 proteins migrate between the 75- and 50-kDa markers. M; molecular weight marker. (B) Schematic representation of the Upf1 deletion mutants used. The numbers denote the amino acid positions relative to the N terminus of human Upf1. The gray boxes indicate a proline/glycine (PG)-rich region, zinc (Zn) finger domains and a serine/glutamine (SQ)-rich region. The dark gray boxes indicate conserved RNA helicase motifs. The ability to interact with Stau2 is indicated on the right. (C) Interaction between Upf1 deletion mutants and Stau2. Each Upf1 deletion mutant fused to GST was transiently co-expressed in 293F cells along with FLAG-Stau2. The cells were lysed at 24 h after transfection, and glutathione-Sepharose beads were used to pull down GST-Upf1 or its deletion mutants. The precipitates were analyzed by Western blotting using anti-GST or anti-Stau2 antibodies. Five percent of the total lysate (upper panels) and 30% of the precipitate (lower panels) were loaded. (D) Schematic representation of the Stau2 deletion mutants used. The numbers denote the amino acid positions relative to the N terminus of rat Stau2. The gray boxes indicate conserved dsRBDs. The ability to interact with Upf1 is indicated on the right. (E) Interaction between Upf1 and the Stau2 deletion mutants. GST-Upf1 was co-expressed in 293F cells with each GFP-fused Stau2 deletion mutant. The interaction was analyzed in the same way as in (C). The positions of the full-length and the deletion mutant proteins are indicated by asterisks.