Covalent modification of MEKK1 by Bio-ITC is blocked by preincubation of cells with PEITC. CV-1 cells expressing wild type MEKK1 or the C1238V mutant were either left untreated or incubated with 250 μM PEITC for 20 minutes at 37°C. Lysates were prepared in TLB, clarified and then 500 μM Bio-ITC was added to the samples as indicated. MEKK1 proteins were purified from the lysates on chitin beads, and either electrophoresed for detection of biotin labeling using streptavidin-HRP (top panel) or assayed for kinase activity (middle panel). To confirm equivalent protein, the SA-HRP blot was re-probed with anti-MEKK1 as in Figure 5. Lane 2 contains the molecular weight markers. Bio-ITC covalently modifies MEKK1 protein and inhibits its activity in a manner that requires C1238V. The modification by Bio-ITC is completely inhibited by preincubation of the cells with PEITC, suggesting that the modification site is completely occupied by the natural isothiocyanate after exposure of intact cells.