Figure 2. The APRO domain of BTG2 interacts directly with the first RRM domain of PABPC1.

(a) Organization of the human PABPC1 protein. The RRM and C-terminal MLLE domains are highlighted. Amino-acid positions relevant for this study are shown. (b) Interaction of BTG2, BTG1 and Tob1 APRO domains with CAF1 (CNOT7 paralogue) and the first RRM domains of PABPC1 in yeast two-hybrid assay. Interaction between the different chimeric proteins indicated was monitored by β-galactosidase assays. Activities are expressed in arbitrary units (a.u.). Experiment was performed twice with two biological duplicates. Error bars (s.d.) correspond to two biological replicates. (c) Co-purification experiments showing direct interaction between BTG2(APRO) and the first RRM domain of PABPC1. GST- and His-recombinant proteins were co-expressed in E. coli as indicated. His-tagged proteins were purified with Nickel beads. Proteins were resolved on 15% SDS–PAGE gels and stained with Coomassie blue. (d) Co-immunoprecipitation of PABPC1 with BTG2 APRO domain. HEK293 cells were transfected with plasmids expressing GFP-BTG2(APRO)-HA or GFP alone. Twenty-four hours after transfection, cells were treated with Dithio-bis Succinimidyl Propionate 0.25 mM and lysed in the presence of RNAse A. Proteins were then precipitated with GFP-trap beads and revealed by western blot with anti-GFP and anti-PABPC1 antibodies. Three biological replicates were performed, two being shown.