STAT5 dimerization requires acetylation. (A) Mass spectra of acetylated peptides recovered from STAT5b prepared from 293T cells transfected with STAT5 and CBP. (B) STAT5b WT and STAT5b with a K→R mutation were transiently transfected along with CBP in 293T cells. Immunoprecipitated (IP) STAT5b proteins were analyzed with pan–anti-acetyl-lysine. (C) STAT5 acetylation in T47D cells received PRL treatment for different times and was analyzed with specific polyclonal antibodies against ak359-STAT5b, aK694-STAT5b, and aK701-STAT5b, respectively. (D) In T47D, CBP down-regulation with siRNA on STAT5 acetylation (pan–anti-acetyl-lysine) in response to PRL treatment for 30 min. (E) In PC3 cells, Myc-tagged and HA-tagged STAT5b (WT or Lysine-to-Arginine (KR) mutant) was cotransfected, followed by treatment with or without PRL for 30 min. Anti-Myc immunoprecipitates were blotted with anti-HA. (F) In PC3 cells, empty vector (EV), STAT5b WT, or STAT5b KR mutants were transfected along with pLHRE-luciferase reporter, followed by treatment with PRL for 6–12 h before luciferase activity assay. (G) Myc-STAT5b was transfected along with EV, PRLR WT, or PRLR K1–15R in 293T cells, followed by PRL treatment for 30 min. Anti-Myc immunoprecipitates were analyzed with indicated antibodies. (H) 293T cells were transiently transfected with PRLR and JAK2, followed by PRL treatment for 60 min. Immunoprecipitated JAK2 was analyzed for PRLR association or phospho-JAK2. (I) (Left) In 293T cells, PRLR WT and indicated PRLR mutants were transfected along with pLHRE-luciferase reporter, followed by PRL treatment for 6–12 h before luciferase activity assay. In 293T cells, EV, CBP, or SIRT2 was cotransfected with PRLR and luciferase reporter or siRNA of control (CTL), CBP, or SIRT was cotransfected with PRLR and luciferase reporter. (Right) Luciferase activity assay was performed under the same conditions as above.