HEK293 cells were grown in RPMI supplemented with 10% FBS at 37°C in 5% CO2. HEK293 cells were transfected with HA–TEAD2 and/or Myc–VGLL3, as indicated. The tags were introduced to the 5′ end through modification of the pCI-neo vector (Promega). Constructs were transfected using Lipofectamine 2000. At 24 h post transfection, the cells were lysed in buffer with 20 mM Tris-HCl pH 8.0, 150 mM NaCl and 0.1% Triton X-100 and protease inhibitors. HA–TEAD2 was immunoprecipitated using anti-HA beads (Sigma) and the co-immunoprecipitated YAP was detected using anti-YAP antibody (D8H1X, Cell Signaling). HA–TEAD2 was detected using anti-HA-HRP (Sigma) and Myc–VGLL3 was detected using clone 4A6 antibody (Millipore). For full details of antibodies, see Table S8 .
Labelled human YAP (amino acids 61–100) and unlabelled human VGLL3 (amino acids 86–112) peptides were used. Histidine-tagged human TEAD4 (amino acids 217–434) was expressed in E.coli and purified by using immobilized metal affinity chromatography (IMAC) and gel filtration chromatography. TEAD4 was incubated with an increasing concentration of VGLL3 peptide prior to the addition of labelled YAP. After a 10-min incubation at room temperature, a native gel (10%) was run at 100 V to visualize free and TEAD-bound YAP.
Labelled human YAP (amino acids 61–100) and unlabelled human VGLL3 (amino acids 86–112) peptides were used. Histidine-tagged human TEAD4 (amino acids 217–434) was expressed in E.coli and purified by using immobilized metal affinity chromatography (IMAC) and gel filtration chromatography. TEAD4 was incubated with an increasing concentration of VGLL3 peptide prior to the addition of labelled YAP. After a 10-min incubation at room temperature, a native gel (10%) was run at 100 V to visualize free and TEAD-bound YAP.