Fig. 3

ABF induces YAP phosphorylation at multiple sites. a The ABF-induced shifting of YAP was caused by YAP phosphorylation. Following treatment with ABF (20 nM), cell lysates were collected and evaluated by coimmunoprecipitation and Western blotting in the presence or absence of alkaline phosphatase. b Quantification of the shifting of YAP, as described above. Data represent the mean ± SEM, n = 3, ^***P < 0.001 versus the control. c Levels of p-YAP (Ser109), p-YAP (Ser127) and p-YAP (Try357) in MCF-7 cells treated with ABF. d Quantitative data of the indicated protein. Data represent the mean ± SEM, n = 3. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 versus the control. e Identification of YAP phosphorylation sites. Following treatment, the phosphopeptides were enriched by Ti⁴⁺-IMAC chromatography. Potential YAP phosphopeptides were analyzed by the LC–MS/MS system. The peptide sequences of YAP recovered by mass spectrometry are labeled in black, the reported phosphorylation sites are labeled in green and the newly identified phosphorylation sites are labeled in red