LATS1/2 kinase activity assay

To assess LATS1/2 enzyme activity, immunoprecipitation followed by western blotting of phosphorylated YAP1 was performed. Muscle specimens were lysed in 1% Nonidet P-40 lysis buffer supplement containing 1 mM DTT and 1× phosphatase inhibitor (Sigma-Aldrich). For immunoprecipitation-kinase assay, 100 μg of protein lysate were mixed with 2 μg of monoclonal antibody against YAP1 (1:400) (Santa Cruz Biotechnology, Inc.) together with Protein G beads and incubated at 4°C for 3 h. Then, beads were washed twice with 1% Nonidet P-40 lysis buffer, 1 mM DTT; once for 10 min with 1% Nonidet P-40 lysis buffer, 500 mM NaCl, 1 mM DTT at 4°C; and twice with 20 mM Tris-HCl (pH 7.4), 1 mM DTT, 1× phosphatase inhibitor. The washed beads were mixed with 2 μg of YAP-GST substrates in a kinase buffer (20 mM Tris-HCl (pH 7.5), 5 mM MgCl2, 5 mM MnCl2, 1× phosphatase inhibitor, 2 mM DTT, 10 μM ATP, 5 μCi of [γ-32P]ATP) and incubated at 30° C for 30 min. The reaction was stopped by adding 7 μl of 5× SDS sample dye, boiled at 100°C for 5 min, and subjected to SDS-PAGE. After electrophoresis, the proteins were transferred to a nitrocellulose membrane, and exposed to autoradiograph film for 0.5–2 h. to test phosphorylation of YAP1 by LATS1/2.