Determination of phosphorylation targets within p300. (A) Schematic representation of domain structure of wild-type p300 and deletion mutants used in panels B and C. Four regions rich in proline-directed serine/threonine-rich residues are denoted STP1 to STP4. (B,C) Deletion variants of FLAG-p300 lacking single STP regions (B) or 3 STP regions (C) were transiently expressed in HEK293 cells in the presence or absence of exogenous RUNX1/PEBP2-β, as indicated. Cells were lysed 24 hours after transfection and the relative phosphorylation efficiencies of the p300 variants were analyzed by Western blotting using anti-FLAG antibodies. (D) The amino acid sequences of STP2 region (residues 828-889 of p300). Proline (P)-directed serine (S) and threonine (T) residues targeted in mutants p300^Δ1,3,4/m1-m3 are changed to alanine, as denoted by asterisks. (E) Transient transfection and Western blot analyses of p300 mutants in HEK293 cells. Wild-type and point mutants of p300^ΔSTP1,3,4 proteins were coexpressed with RUNX1/PEBP2-β as illustrated and analyzed by Western blotting as described in panel B.