ROCKI is phosphorylated at Ser1333 residue. (A) The flag-ROCKI protein was expressed in HEK293T cells and immunoprecipitated with anti-flag antibody. Equal amounts of immunoprecipitated complex were pre-incubated with 100 μM of Y27632 for 20 min and subjected to an in vitro kinase reaction with 5 μCi of [γ-32P] ATP. (B) Wild-type (WT) and S1333A mutant of flag-ROCKI proteins were immunoprecipitated for in vitro kinase reaction. After 20 min, the reaction was stopped and proteins were then applied to SDS-PAGE followed by autoradiography. The protein level of flag-ROCKI was determined by Western blotting with anti-ROCKI antibody.