Cultured human embryonic kidney 293 cells were transfected with flag-MEKK3 and HA-MEK5 plasmids, and cells were lysed with radioimmunoprecipitation assay buffer 48 hours after transfection. The lysates were subject to pulldown with anti-flag antibody affinity gel (Sigma-Aldrich). After washing, protein bound to the beads were eluted and subjected to Western blot analysis.

For in vitro kinase assays, purified GST-MEK5 was used as the substrate. Purified flag-MEKK3 and GST-MEK5 were incubated in the kinase reaction buffer system [20 mM Hepes/KOH (pH 7.5), 5 mM MgCl2, 2 mM dithiothreitol, 1 mM ATP] for 4 hours at 30°C. The reaction mixtures were then denatured with Laemmli loading buffer, and Western blots were performed using a standard protocol. Phosphorylated MEK5 was detected with the p-MEK5 antibody from Novus.

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