FOXO1 Interacts with SMAD3/4.

A. GST interaction assays were performed using bacterially expressed GST-fusion proteins (indicated above each lane) and ³⁵S-labeled in vitro transcribed and translated GFP, FOXO1, FOXO1-CA, and FOXO1-CA-DBD mutant (indicated on the left of the panel). GFP was used as a negative control. The GST-fusion proteins included GST alone, GST-SMAD2 (S2), GST-SMAD3 (S3), and GST-SMAD4 (S4). One quarter of the protein used in the interaction assay was loaded in the lane marked input. The experiment was repeated several times with similar results and a representative experiment is shown. B. Co-immunoprecipitation assays were performed using nuclear extracts from LβT2 cells treated with or without 10 ng/mL activin for 2 h after an overnight incubation in serum-free media. One tenth of the protein used in the immunoprecipitation reaction was loaded in the lane marked input. The immunoprecipitation was performed with mouse IgG, SMAD4 (S4) or SMAD2/3 (S2/3) antibody, as indicated. Western blot analysis was performed using SMAD4, SMAD2/3 and FOXO1 primary antibodies, and a horseradish peroxidase-linked secondary antibody. The experiment was repeated several times with similar results and a representative experiment is shown.