Determination of the binding site of TEAD to YAP and vice versa. (a) Overlapping dodecapeptides with two amino acid shift covering the whole human TEAD proteins were bound to a solid support. The membrane was incubated sequentially with YAP-GST protein, and anti-GST antibody, followed by a peroxydase-labeled secondary antibody. The membrane was revealed with ECL system. The sequence corresponding to the identified spots is shown. (b) Overlapping dodecapeptides with two amino acids shift covering the YAP loop described as binding area to TEAD were synthesized and bound to a solid support. The membrane was incubated with TEAD-GST protein, followed by anti-GST antibody and a secondary peroxidase-conjugated antibody. The membrane was revealed using the ECL system. The sequence corresponding to the identified spots is shown. (c) Competition of TEAD/YAP interaction was done in vitro using purified proteins and a concentration or 250 μM of TEAD or irrelevant peptide PfMut3 DPT-LRR1.1. Competition was done for 1 h at room temperature. Immunoprecipitates were washed and immunoblotted with anti-YAP antibody and anti-TEAD antibody, as internal control. Densitometry of the protein bands and ratio calculation is also shown. (d) Competition of TEAD/Yap was also analysed upon membrane hybridation. Yap-GST protein was pre-incubated with TEAD peptide for 1 h at room temperature. The membrane was incubated with the mix Yap-GST protein/TEAD peptide. After washing steps, the membrane was incubated with anti Yap antibody followed by a peroxiydase-labelled secondary antibody. The membrane was revealed using the ECL system.