Figure 1

Identification of Stau2-interacting proteins. (A) A portion (nucleotides 396-907) of the human Stau2 cDNA (GenBank ID: NM_001164381) was amplified by RT-PCR from total RNA extracted from 293F or HeLa cells. The amplification was performed with (+) or without (-) a reverse transcription (RT) step. The products were analyzed by agarose gel electrophoresis followed by ethidium bromide staining. M; molecular weight marker. (B) 293F or HeLa cells were transfected with a negative control siRNA (siNC) or one of two different siRNAs to deplete Stau2 (siStau2-1 and siStau2-2). The cells were harvested at 48 h after siRNA transfection, lysed, and subjected to Western blotting using anti-Stau2 and anti-GAPDH antibodies. The positions of the Stau2 protein isoforms are indicated by asterisks. (C, D) GST-Stau2 and GST were precipitated from 293F-GST-Stau2 and 293F-GST cell extracts, respectively, with glutathione-Sepharose beads. After extensive washing, the bound proteins were eluted with reduced glutathione and subjected to SDS-PAGE. The protein bands were visualized by silver staining (C) and Western blotting using anti-GST, anti-RHA, anti-Upf1 and anti-Mov10 antibodies (D). The three specific bands migrating between 150 and 100 kDa were identified as RHA (142 kDa), Upf1 (124 kDa) and Mov10 (114 kDa) by mass spectrometry. M; molecular weight marker. (E) Immunoprecipitation from HeLa cell extracts was performed using a rabbit IgG or a rabbit anti-Stau2 antibody. Two percent of the total cell lysate and 10% of the precipitate were subjected to SDS-PAGE followed by Western blotting. HC; IgG heavy chain.