Identifying the post-translational modifications of the MEKK1 protein.

<p><i>A</i>. The PHD mutant can be phosphorylated. Vector, wild type flag-MEKK1 and flag-PHD mutant (mutP) were transfected into 293T cells and immunoprecipitated with αflag M2 conjugated beads. Membranes were probed with total or phospho-MEKK1. <i>B</i>. CIP treatment does not affect basal MEKK1 modification, but does shift the molecular weight of vinblastine treated (phosphorylated) endogenous MEKK1. DT40 cells were treated with vinblastine for six hours and lysates were immunoprecipitated with αMEKK1. Half of each lysate was treated with calf alkaline phosphatase and all were incubated at 37°, run on SDS-PAGE gel and membranes were probed with αMEKK1. <i>C</i>. Inhibition of de-ubiquitinating enzymes via N-ethylmaleimide (NEM) stabilizes the higher molecular weight form of MEKK1. Wild type DT40 cells were lysed in modified RIPA. Lysates were treated with or without 20 mM NEM for 30 minutes at room temperature. Lysates were run on SDS-PAGE and probed with α MEKK1. <i>D</i>. MEKK1 is ubiquitinated whereas the PHD mutant is not. 293T cells were transiently transfected with vector, flag-MEKK1 or mutP. Cell lysates were immunoprecipitated with flag-conjugated beads, run on an SDS-PAGE gel and probed with αubiquitin or αMEKK1.