FIG. 2.

(A) Binding of JSAP1 to mammalian MAPKs. COS-7 cells were transiently cotransfected with 0.4 μg of pFlag-CMV2-JNK1, -JNK2, -JNK3, -p38α or -ERK2 along with 1.1 μg of either pcDNA3-His-S empty vector or pcDNA3-His-S-JSAP1. Cell lysates were precipitated with S-protein-linked agarose (S-PA) and analyzed by immunoblotting with anti-Flag antibody. Expression of Flag-MAPKs and His-S-JSAP1 was examined by immunoblotting 1/10 of the cell lysates used in the binding reactions. (B) Mapping of the JNK3-binding region on JSAP1. In vitro-translated ³⁵S-labeled JNK3 was incubated with 0.5 μg of either immobilized Trx-His-S protein or Trx-His-S-tagged segments of JSAP1. The protein complexes were extensively washed, and the bound JNK3 was detected by SDS-PAGE and autoradiography. One-tenth of the ³⁵S-labeled protein used in the binding reactions was loaded as a positive control.