ERK has higher affinity for ERG than ETS1 and ERG is phosphorylated in cells with lower pERK levels. (A, B) A transwell assay compared cell migration of RWPE1 and RWPE-KRAS (RWPE2) cell lines over-expressing the indicated ETS protein compared to empty vector. Mean and SEM of three biological replicates are shown. P-values (* < 0.01, ** < 0.005) by T-test. (C) Indicated amount of purified ETS1, ETS2 and ERG were phosphorylated in vitro by ERK2 kinase. Coomassie staining indicates total protein and an autoradiograph, phosphorylation. (D) Quantification of radiolabel density in (C) in arbitrary density units. (E) The binding of fluorescently labeled ERK2 to varying concentrations of ERG or ETS1 by microscale thermophoresis (MST). (F) Immunoblots with indicated antibodies of cell extracts or Flag-immunoprecipitate from cells stably over-expressing ERG in the presence of the MEK inhibitor U0126, or over-expressed KRAS, as indicated. (G) Immunoblots as in F, but with cells stably over-expressing ETS1.