Phospho site mapping of Pim phosphorylation sites in Bad. Bad was phosphorylated by 10 mU of Pim-1 (A), Pim-2 (B) or Pim-3 (C) at 30°C for 10 min. Bad was then digested with trypsin as described in the methods and the resulting peptides resolved using an acetonitrile gradient on reverse phase HPLC. Phospho peptides were analysed by ms/ms and solid phase Edman sequencing to determine the phosphorylated residue. (D) Solid phase sequencing results for the peaks obtained with Bad phosphorylated by Pim-3. For each peptide, release of 32P was obtained within the first 7 cycles (see section D). Further cycles to the end of the peptides did not result in the release of more peaks of 32P (data not shown). Similar results were obtained for peaks 1 to 4 using Pim-1 and Pim-2 phosphorylated Bad (data not shown).