Imatinib is a potent inhibitor of PDGF-induced Akt and Crk-L activation in osteoblast cells. (A) Bone trephine explant cultures were serum deprived, treated with or without imatinib (3 μM), then stimulated with PDGF-BB (10 ng/mL) for the indicated times, after which cell lysates harvested. Equivalent levels of cellular proteins were transferred to PVDF membranes and probed with phosphorylation-specific antibodies as indicated. (B) To determine the concentration range at which imatinib was inhibitory to PDGF-induced p38, Akt, and Crk-L phosphorylation, serum-deprived cells were treated with a range of imatinib concentrations, stimulated with PDGF (10 ng/mL) for 10 minutes, and then processed as above. Western blots were probed with phosphorylation-specific antibodies as indicated. Untreated lysates are labeled UT. (C) To determine whether imatinib had a sustained effect on p38, Akt, and Crk-L activation, lysates were harvested from osteogenic cultures treated with or without 3 μM imatinib at weekly intervals for 2 weeks. Proteins were transferred to PVDF membranes and probed with phosphorylation-specific antibodies as indicated.