FIGURE 9.
Active variants of Erks are also spontaneously active in mammalian cells. The cDNAs encoding the specified mutants were introduced into HEK293 cells. Erk1 proteins were fused to a poly-His tag, and Erk2 proteins were fused to a HA tag. 24 h after transfection, cells were harvested. Erk molecules were immunoprecipitated from lysates, and immunoprecipitates were subjected to a kinase assay without a substrate. Assay mixtures were separated on SDS-PAGE and transferred to nitrocellulose membranes that were exposed to x-ray film. The upper panel shows the autoradiograms. The lower panels show Western blots (WB) of the same nitrocellulose membranes, using anti-phosphorylated-Erk (p-Erk), anti-HA, and anti-His antibodies.