PIM proteins were immunoprecipitated from MV4;11 cells and the agarose-protein A-immunoprecipitate complex was tested for its ability to phosphorylate BAD in vitro in the presence or absence of K00135. Phosphorylation of BAD (both on Ser112 and Ser136, detected by WB with phospho-specific antibodies) was abrogated on addition of the compound. Asterisks, strong bands corresponding to the heavy chain of the anti-PIM2 rabbit antibody recognized by the antirabbit immunoglobulin G secondary antibody. Beads alone (without anti-PIM antibodies) were incubated with the MV4;11 extract and used for the same in vitro phosphorylation reaction as a negative control.
