Figure 2.

Identification of 14-3-3 binding site on MDMX. (A) Location of phosphorylation sites on MDMX. Phosphorylation of S342, S367, and S403 was detected by mass spectrometry and verified by phosphorylation-specific antibody analyses in previous studies. (B) MDMX mutant binding to 14-3-3. H1299 cells were transiently cotransfected with MDMX mutant and FLAG-14-3-3τ plasmids for 48 h, followed by anti-FLAG IP and MDMX Western blot. (C) 14-3-3 binding by phosphorylated S367 peptide. MDMX peptides with or without phosphorylation on S342 and S367 were chemically crosslinked to agarose beads. The beads were incubated with whole-cell extract, washed, and bound 14-3-3τ detected by Western blot.