Figure 2. c-Abl Enhances MST2 Kinase Activity.

(A). Lysates of Neuro2A cells stably transfected with c-Abl RNAi #1 or #2 or the control vector were immunoblotted with the indicated antibodies. (B). Lysates of HEK 293T cells transfected with FLAG-tagged MST2 alone or together with increasing amounts of Myc-tagged c-Abl expression plasmid were analyzed by immunoblotting with the indicated antibodies. (C). Anti-Myc immunoprecipitates from cells transfected with Myc-c-Abl or the control vector were subjected to the in vitro kinase reaction using the recombinant GST-MST2 or GST alone as substrate. GST-MST2 or GST from phosphorylation reactions was then subjected to the second in vitro kinase assay using GST-FOXO3-FD as substrate. Phosphorylation reactions were analyzed by immunoblotting with anti-pS207-FOXO3 antibody. The experiments were repeated for three times and quantative density is indicated. (D). Lysates of HEK 293T cells transfected with the FLAG-MST2 or–Y81F expression plasmid were immunoprecipitated with the anti-FLAG antibody and subjected to an in vitro kinase assay using Histone H2B as substrate in the presence of [³²P] ATP. Phosphorylation reactions were analyzed by electrophoresis and autoradiography. The experiments were repeated for three times and quantative density is indicated.