Ei24 coprecipitates and colocalizes with specific IMPs and shares homology with the IBB domain of IMPα2. (A) FLAG-Ei24 expressed in HEK293T cells was immunoprecipitated using anti-FLAG (lane 1) or anti-Ei24 (lane 2) antibodies. Bound proteins were separated by SDS-PAGE, transferred to a nylon membrane, stained with a Sypro Ruby dye, and subsequently analyzed by MALDI-TOF MS. Bands identified as IMPβ1, IMP7, and Ei24 are indicated (see Table S1 for details). (B) Endogenous Ei24 or IgG immunoprecipitates (IP)/input lysates from HEK293T cells were resolved by SDS-PAGE before Western analysis using the specific antibodies indicated. (C) Multiple sequence alignment of the human and mouse Ei24 IBBL domains (predicted to form an α-helical structure using Protein Homology/Analogy Recognition Engine version 2.0) together with IMPα2/CanRch1, performed as described in Materials and methods. Numbers indicate the portion of the amino acid residues (single letter code) within the respective proteins. Gray and black shading indicates similar and identical residues, respectively. Sites of targeted mutation in this study are indicated by asterisks. (D) HeLa cells treated with 50 µM etoposide or DMSO vehicle control for 16 h were fixed and immunostained using specific antibodies for endogenous IMPβ1, IMPα2, IMP7, or Ei24, and counterstained with DAPI. Merged images are shown at higher magnification (high mag., bottom panels). Yellow coloration in merged images indicates colocalization; quantitative analysis is presented in Fig. S1, C and D. Bars, 20 µm.