BioITC inhibits MEKK1, but not the C1238V mutant, by covalent modification. A. Purified MEKK1 or the C1238V mutant on chitin beads were treated with Bio-ITC at the concentrations indicated in 50 mM Tris pH7.8 for 40 minutes at room temperature. Bio-ITC was removed, and the kinase activity measured. Bio-ITC inhibits wild type MEKK1, but not the C1238V mutant in a dose dependent manner. B. Purified wild type MEKK1 or the C1238V mutant were treated as in Panel A with Bio-ITC at the indicated concentrations. The treated protein was used to either assay for kinase activity (top panel) or to detect covalent modification by Bio-ITC using a streptavidin-HRP conjugate (middle panel). Equivalent protein was confirmed by re-probing the streptavidin gel with anti-MEKK1 (bottom panel), using an alkaline phosphatase conjugated secondary antibody and colorimetric detection reagents. Bio-ITC inhibited wild type MEKK1 by stable, covalent modification in a manner that requires C1238V. C. CV-1 cells expressing either wild type MEKK1 or the C1238V mutant were lysed in Tris lysis buffer containing 0.1% NP-40. Clarified lysates were treated by addition of either PEITC (P) or Bio-ITC (B) to final concentration of 250 μM, and incubated at room temperature for 30 minutes. MEKK1 proteins were purified on chitin beads and kinase activity measured. Both PEITC and Bio-ITC completely inhibited wild type MEKK1, but not the C1238V mutant when added to cell lysates.