FIGURE 3.

ANG II induces rapid increase in the phosphorylation of YAP at Ser¹²⁷ and Ser³⁹⁷ in IEC-18 cells. A and B, confluent cultures of IEC-18 cells were incubated without (−) or with ANG II (ANG) for the indicated times (A) or concentrations (B). Cultures were then lysed with 2× SDS-PAGE sample buffer and analyzed by immunoblotting with antibodies that detect YAP phosphorylated at Ser¹²⁷ and Ser³⁹⁷. Immunoblotting for total YAP and GADPH was also included. C, quantification of total YAP phosphorylated at Ser¹²⁷ and Ser³⁹⁷ was performed using MultiGauge version 3.0. The results represent the mean ± S.E., n = 3, and are expressed as percentage of the maximal level of YAP phosphorylated at Ser¹²⁷ and Ser³⁹⁷. Similar results were obtained in three independent experiments. D, confluent cultures of IEC-18 cells were stimulated with 10 nm ANG II for 30 min. Then the cells were lysed, and YAP immunoprecipitates (IP: YAP) were analyzed by Western blotting (W.B.) with YAP Ser¹²⁷ and Ser³⁹⁷. E, cultures of IEC-18 cells were transfected with non-targeting siRNA (N. Targ) or with siRNAs targeting YAP for 4 days. The cultures were then stimulated with 10 nm ANG II for 30 min. Cells were lysed with 2× SDS-PAGE sample buffer and analyzed by immunoblotting antibodies that detect YAP phosphorylated at Ser¹²⁷ and Ser³⁹⁷, total YAP, and GAPDH. Similar results were obtained in four independent experiments.