FIG. 6.

CrmA and p35 inhibit the generation of a MEKK1-derived kinase-active cleavage product. Cells were transfected as described in the legend to Fig. 5. (A) Western blot analysis of lysates with antibodies 12CA5 and 95-012. The immunoreactive proteins were detected by ECL. Fragments A, B, C, and D correspond to MEKK1 cleavage products, and the band marked with an asterisk may correspond to a dimer of fragment D (see the text). (B) A kinase-inactive MEKK1 mutant (MEKK1 K→M) is not cleaved into fragments B and C. Cells were transfected with 1 μg of vector alone (pcDNA3), HA-tagged MEKK1 in pcDNA3 (plasmid MEKK1.dn3), or HA-tagged MEKK1 K(1253)→M in pcDNA3 [plasmid MEKK1(−).dn3]. At 18 h after transfection, the cells were lysed and the presence of MEKK1 species was detected by Western blot analysis with antibody 12CA5. The positions of fragments A and B and full-length MEKK1 are indicated. (C) In vitro SEK1 K→M phosphorylation assay performed on cell lysates immunoprecipitated with the indicated antibodies. The positions of MEKK1, fragments C and D, and SEK1 K→M are indicated.