Nuclear pellets were collected and lysed with NP-40 lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP-40, 0.1% Triton X-100. 1 mM PMSF and 1x protease inhibitor were freshly added.). Sonication was performed for 3 minutes with Q800R sonicator (QSonica) at 4°C to improve lysis before centrifuging to collect nuclear lysate. The nuclear lysate was precleared using 10 μl Dynabeads Protein G (Life Technologies) and antibodies were added to lysate and incubated at 4°C overnight. The protein complex was collected using 20 μl Dynabeads Protein G and washed 6 times with NP-40 lysis buffer before elution with 2x SDS loading buffer. The antibodies used in Co-IP were: anti-ERα (sc-8005, Santa Cruz), anti-TEAD4 (sc-101184, Santa Cruz), anti-YAP1 (14074S, Cell Signaling Technology), anti-HA (ab9110, Abcam) and IgG (ab171870, Abcam).
To study the interaction between ERα and wildtype or DNA-binding domain mutant TEAD4, we used two established doxycycline-inducible stable cell lines expressing BLRP and Flag dual-tagged wildtype or mutant TEAD4 proteins (see the section of “Establishing stable cell lines expressing BLRP-Tagged TEAD4 and biotin ChIP ”). The nuclear lysate was prepared and ERα protein complex was pulled down using ERα (sc-8005, Santa Cruz) antibody. The wildtype or mutant TEAD4 proteins were detected with anti-Flag antibody (F1804, Sigma).
To study the interaction between ERα and wildtype or DNA-binding domain mutant TEAD4, we used two established doxycycline-inducible stable cell lines expressing BLRP and Flag dual-tagged wildtype or mutant TEAD4 proteins (see the section of “