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. 2012 Apr 4;287(22):18398–18407. doi: 10.1074/jbc.M111.326538

FIGURE 4.

FIGURE 4.

JNK physically associates with and phosphorylates Raptor. A, HA-Raptor or FLAG-JNK1 was expressed in HEK293 cells. The lysates were immunoprecipitated (IP) with anti-FLAG and immunoblotted with Raptor and FLAG antibodies. WCL, whole-cell lysates. B, the presence of JNK in Raptor immunoprecipitates was determined by immunoblotting with JNK antibody (IP ab). C, the presence of Raptor in JNK immunoprecipitates was determined by immunoblotting with Raptor antibody (IP ab). D, purified GST or GST-JNK1 was incubated with HEK293 lysates prepared using Triton X-100 detergent, and then the GST pulldown assay was performed, and bound Raptor was detected by immunoblotting with Raptor antibody. The amount of GST or GST-JNK1 was shown with Ponceau S staining. E, immunoprecipitated FLAG-Raptor WT or S696A/T706A/S863A (3A) was incubated with purified activated JNK1 in a kinase reaction and then subjected to SDS-PAGE and immunoblotting with phospho-Raptor (p-Raptor) antibodies.