HEK239FT cells were transfected with different vectors: pPGS-3HA-TEAD1, pCMV-Flag-YAP-S94A and pCMV-Flag-S127A-YAP were gifts from Kunliang Guan (Addgene plasmids #33055, #33102 and #27370; RRID: Addgene-33055, -33102 and -27370).
24 h after transfection, media were changed with fresh DMEM containing 1% FCS for 24 h. Transfected cells were then rinsed with ice-cold PBS and lysed in IP-lysis buffer (Invitrogen, Courtaboeuf, France). Equal amounts of proteins were precleared overnight at 4 °C using Protein-A/G-agarose (Santa Cruz Biotechnology, CA, USA). Supernatants were incubated with primary antibody against Flag (Sigma Aldrich) and HA-tag (Cell signaling), for 2 h at 4 °C. 50 µL of Protein-A/G-agarose was then added and incubated overnight at 4 °C. Beads were washed three times with IP-lysis buffer; thereafter, 30 µL of Laemmli buffer was added and boiled for 5 min. After centrifugation, supernatants were harvested and processed for SDS-PAGE and Western blot as described above.
24 h after transfection, media were changed with fresh DMEM containing 1% FCS for 24 h. Transfected cells were then rinsed with ice-cold PBS and lysed in IP-lysis buffer (Invitrogen, Courtaboeuf, France). Equal amounts of proteins were precleared overnight at 4 °C using Protein-A/G-agarose (Santa Cruz Biotechnology, CA, USA). Supernatants were incubated with primary antibody against Flag (Sigma Aldrich) and HA-tag (Cell signaling), for 2 h at 4 °C. 50 µL of Protein-A/G-agarose was then added and incubated overnight at 4 °C. Beads were washed three times with IP-lysis buffer; thereafter, 30 µL of Laemmli buffer was added and boiled for 5 min. After centrifugation, supernatants were harvested and processed for SDS-PAGE and Western blot as described above.