LN229 cells were harvested, lysed, and immunoprecipitated with anti-YAP1 (MAB8094, RD, USA) or IgG conjugated to protein G agarose (11243233001, Roche, USA) at 4 ºC overnight. Then, the immunoprecipitates were separated on SDS-PAGE gels. After coomassie blue staining, the protein bands were collected for mass spectrometry analysis.

The monolayer cells or 3D cells were lysed in the IP lysis buffer (pH 7.4, 25 mM Tris, 150 mM NaCl, 1 mM EDTA, 1% NP40, 5% glycerol, P0013, Beyotime, China). The proteins were immuno-precipitated with anti-YAP1 (MAB8094, RD, USA) or IgG conjugated to protein G agarose (11243233001, Roche, USA) at 4 ºC overnight. Subsequently, the immuno-precipitates were used for Western blotting analysis following the procedure described in our previous study 31. The antibodies used were against human TEAD4 (ab151274, Abcam, Cambridge, UK) and YAP1 (ab52771, Abcam, Cambridge, UK). β-actin (ab179467, Abcam, Cambridge, UK) was used as the protein loading control.

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