FIG. 2.

Selective binding of JIP1 and JIP2 to the mixed-lineage group of MAPKKKs. (A) Epitope-tagged JIP1 or JIP2 was expressed in cells together with epitope-tagged MAPKKKs. Control experiments were performed by transfection of the empty expression vector instead of the JIP expression vector. The expression of JIP and MAPKKK proteins was examined by immunoblot analysis of the cell lysates. The MAPKKK proteins were immunoprecipitated with the M2 monoclonal antibody to the Flag epitope. The presence of JIP proteins in the immunoprecipitates (IP) was examined by immunoblot analysis using a monoclonal antibody that binds the T7-Tag epitope. Coimmunoprecipitation was not observed in experiments using MEKK1, MEKK3, or MEKK4. In contrast, JIP1 and JIP2 coimmunoprecipitated with the MLKs DLK, MLK2, and MLK3. (B) The COOH-terminal regions of JIP1 (residues 283 to 660) and JIP2 (residues 499 to 824) were expressed in cells as GST fusion proteins together with epitope-tagged MLK. Control experiments were performed by transfection with the GST expression vector pEBG. The amounts of the GST proteins and MLK in the cell lysates were examined by protein immunoblot analysis. The GST fusion proteins were precipitated from cell lysates with glutathione-agarose, and MLK present in the pellet was detected by protein immunoblot analysis.