Western Blot analysis of PIM2 (arrow) using 040060. 293 cell lysates (2ug/lane) either nontransfected (Lane 1) or transiently transfected (Lane 2) with the PIM2 gene.

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 040060 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.

Formalin-fixed and paraffin-embedded human cancer tissue reacted with 040060, which was peroxidase-conjugated to the secondary antibody, followed by DAB staining.