Msi1 competed with binding of eIF4G to PABP. (A) Illustration of the PABP variants. (B) Flag-Msi1 or Flag-eIF4GN-(1–582) was coimmunoprecipitated with Myc-PABP variants in 293T cells using anti-FLAG resin. Notably, Msi1 and eIF4G bound to a common domain within PABP (middle, bottom). (C) In vitro competition assay between purified GST-Msi1 and purified Flag-eIF4G (45–1560)-His immobilized FLAG resin. The CBB-stained, purified fusion proteins Flag-eIF4G (41–1560)-His, GST-PABP, GST, GST-Msi1-D2, and GST-Msi1 are shown (left panel, lanes 1–5). (D–F) Analysis of the kinetics of PABP's interaction with Msi1 or eIF4G by the QCM. (D) Illustration of the His-tagged proteins immobilized on the QCM plate and GST-PABP. The His-tag proteins were anchored to the QCM plate by an anti-His antibody. (E) Curves showing the time course of the changes in frequency for the proteins coated on the QCM plate, His-eIF4G 41-244mut, His-Msi1-D2, His-eIF4G 41–244, His-Msi1, and Flag-eIF4G (41–1560)-His, in response to the addition of 100 nM GST-PABP. (F) Summary of the kinetics parameters for the binding of PABP to Msi1 or eIF4G on the QCM; for a more detailed description see Materials and methods. (G) In vivo competition assay using 293T cells expressing Flag-eIF4GN and Myc-Msi1. The quantitative analysis was performed with Multigauge software (Fujifilm) in C and G (n = 5, mean ± SEM; *, P < 0.01 vs. control; †, P < 0.05 vs. control).