Phosphorylation of peptides derived from Bad and recombinant Bad protein by Pim kinases. A-C) Recombinant Pim-1 (A), Pim-2 (B) or Pim-3 (C) were incubated in in vitro kinase assays with the indicated concentrations of synthetic peptides from around the three potential phosphorylation sites in Bad as described in the methods. The peptides used were RSRMSSYPDRG (corresponding to Ser112), RGSRSAPPNL (corresponding to Ser136) and RELRRMSDEFEGS (corresponding to Ser155). After 10 min at 30°C, the reactions were terminated and the amount of phosphate incorporated into the peptide determined. D-F) Recombinant Pim-1 (D), Pim-2 (E) or Pim-3 (F) were used to phosphorylate 2 μg of Bad as described in the methods. At the times indicated reactions were stopped by the addition of SDS sample buffer and the amount of phosphate incorporated into Bad was determined by SDS gel electrophoresis and Cerenkov counting of the Bad band.