Identification of PABP as an Msi1-specific binding protein by the TAP method. (A) Msi1-bound proteins that were extracted from 293T cells expressing Flag-Msi1-TAP were resolved by SDS–PAGE, visualized by CBB staining (lanes 2 and 4), and compared with those of control Flag-TAP-expressing cells (lanes 1 and 3). The bound proteins in the TEV-digested extracts are shown in lanes 1 and 2; similarly, those of in the final extracts are shown in lanes 3 and 4. CBB-stained PABP, IMP, and Msi1 are indicated with arrowheads. (B) Msi1 colocalized with PABP and IMP3 in the cytoplasm. P19 cells were stained with anti-Msi1 (green) antibody, and anti-PABP (red, top) or anti-IMP3 (red, bottom) antibodies. Nuclei were stained with Hoechst (blue in P19 cells) in the merged image. (C) Immunoblottings after immunoprecipitation with ant-Msi1 antibody using E14 mouse brain extracts were performed with each antibody, respectively. (D) Protein extracts prepared from mouse brain at E16 were mixed with bacterially expressed and purified GST or GST-PABP fusion proteins. The GST fusion proteins were stained with CBB (lanes 1 and 2). Elutes were analyzed by immunoblotting using anti-eIF4G, anti-Msi1 14H1, or anti-eIF4E antibodies (lanes 3–6). (E) Double-immunohistochemistry of Msi1 (red) and PABP (green), eIF4G (green), or Sox1/(2)/3 (green) in coronal sections of the E14 forebrain. Sox1/(2)/3 is a marker for neural precursor cells. Inset in E shows a low magnification view of the main Msi1-expressing regions. CP, cortical plate; IZ, intermediate zone; VZ, ventricular zone. Bars: 5 μm (B), 50 μm (E).