Importance of S216 for the ETV1:14-3-3 interaction. (A) Sketch of ETV1. AD, activation domain; RD, regulatory domain; ETS, DNA-binding domain. Phosphorylation sites for MAPKs (S94, T139, T143, S146) or MAPKAPKs (S191, S216, S334) are pointed out. (B) 6Myc-tagged truncations of ETV1 as well as wild-type or phosphorylation site mutants of full-length 6Myc-ETV1 were coexpressed with 14-3-3τ in 293T cells. After anti-Myc immunoprecipitation, coprecipitated 14-3-3τ was revealed by Western blotting. (C) HA-tagged 14-3-3τ, but not its R56,60A mutant, coimmunoprecipitated with 6Myc-ETV1 in 293T cells. Asterisk marks HA-14-3-3τ that co-migrated with an unspecific band. IgL, immunoglobulin light chain. (D) RT-PCR analysis of 293T cells transfected with indicated plasmids.