FIG. 6.

Effective JNK1 activation in a multimolecular complex. (A) COS-1 cells were transfected with 0.1 μg of control vector or GST-MEKK2(CT) expression vector per plate, together with 0.5 μg of HA-JNK1 per plate. The cells were lysed 40 h later. Half of the lysates was used to precipitate the MEKK2(CT)-containing complex by adding GSH-Sepharose beads as described above; the other half was used to immunoprecipitate (IP) HA-JNK1 by adding the 12CA5 monoclonal antibody. The amount of HA-JNK1 precipitated by IP and GST-Sepharose beads was determined by Western blotting (WB). Equal amounts of HA-JNK1 from each precipitation were assayed for JNK activity using GST-c-Jun as substrate, as described in the legend to Fig. 1A. The relative fold increases of JNK1 activity are shown. (B) GST-MEKK2CT (10 ng/plate) and HA-JNK1 (0.5 μg/plate) expression vectors were cotransfected into COS-1 cells with expression vectors for HA-JNKK2 (0.5 μg/plate) or HA-JNKK2(AL) (0.5 μg/plate) as indicated. Cells were lysed 40 h later, and GSH-Sepharose beads were added to coprecipitate HA-JNK1 from the lysates as described in the legend to Fig. 4. The coprecipitated HA-JNK1 was assayed for kinase activity as described in the legend to Fig. 1A. Expression levels of HA-JNKK2, HA-JNKK2(AL), and HA-JNK1 were determined by Western blotting (WB). The relative fold increases of JNK1 activity are shown. (C) GST-MEKK2(CT) (0.1 μg/plate) and HA-JNK1 (0.5 μg/plate) expression vectors were cotransfected into COS-1 cells with 0.5 μg of expression vectors for HA-JNKK2 or HA-JNKK2(AL) per plate as shown. The cells were lysed 40 h later, and GSH-Sepharose beads were added to precipitate the MEKK2(CT)-containing complex. Coprecipitated HA-JNKK2, HA-JNKK2(AL), and HA-JNK1 (upper panel) and their expression levels in the cell lysates (lower panel) were determined by Western blotting (WB).