Figure 3.

CUGBP1 inhibits p27 IRES reporter mRNA translation. (A) Recombinant CUGBP1 protein was expressed and purified as described in Methods. CUGBP1 in each elution fraction was detected by western blotting. The purified protein from the 200 mM imdazole elution fraction (lane 3) was dialyzed and further used for the following experiment. (B) Diagram of reporter mRNAs 472-Luc, rev472-Luc and control-Luc used in the following experiment. (C) Reporter mRNAs were synthesized and used for in vitro translation as described in Methods section. Increasing doses of rCUGBP1 protein (1 × 50 ng, 5 × 250 ng) was added in reaction mixture. The translation efficiency of each sample was measured by luciferase assay. (D) Different reporter mRNAs were used for in vitro translation with or without 250 ng rCUGBP1. The translation efficiency of each sample was measured by luciferase assay.