ERG requires S215 and the FXFP motif to promote prostate cell migration and activate target genes. (A, D, G) Flag Immunoblots of RWPE1 cells stably expressing the indicated Flag-ERG proteins. (B, E, H) Representative images of transwell migration assays in the given cell lines (C, F, I) Quantification of transwell assays shows relative number of cells migrated compared to empty vector as the mean and SEM of at least three biological replicates. (J) qRT-PCR measured relative mRNA levels of migration related genes in ERG expressing RWPE1 cells compared to those expressing vector only. Mean and SEM of three replicates. (K) Nuclear (Nu) and cytoplasmic (Cy) fractions of RWPE1 cells expressing the indicated Flag-tagged proteins were immunoblotted with the indicated antibodies. Histone H3 and tubulin control for proper fractionation. All lanes are from the same exposure of the same blot, but extraneous lanes were removed at the vertical line. (L) Chromatin immunoprecipitation (ChIP) of ERG or ERG-S215 at three target loci. Two biological replicates (R1 and R2) are shown. Enrichment is the ratio of the copy number of the target loci to the mean copy number of two negative control loci in the same ChIP sample, as measured by quantitative PCR. All P values in this figure by T-test (* < 0.01, ** < 0.005, *** < 0.001).