The effect of PD166326 on leukemic cell signaling in vivo. (A) Bone marrow protein lysates from mice with the CML-like disease on chronic PD166326 (PD16), imatinib mesylate, or placebo therapy were analyzed by anti-phosphotyrosine immunoblot. A protein size marker (M) is shown at far left. (B) Spleen protein lysates from PD16-, imatinib mesylate–, or placebo-treated mice were subjected to anti–Crk-L immunoblot (IB) as a surrogate marker of Bcr/Abl kinase activity in vivo. The positions of the upper (phosphorylated; p-Crk-L) and lower (Crk-L; nonphosphorylated) forms are shown at right. The ratio of p-Crk-L/Crk-L was quantitated by densitometry and is shown at the top of each lane. Parental or P210-Ba/F3 cells are shown in the bottom panel as a control. (C) The same samples in panel B were immunoprecipitated with anti-Lyn antibody and probed with an antibody that recognizes activated (pLyn) or total Lyn protein. (D) The same samples in panel B were probed with an antibody that recognizes activated (pERK) or total ERK1/2. The average pERK/ERK ratio by densitometry was 0.8 for PD16, 0.5 for imatinib mesylate, and 0.6 for placebo samples. Lysates from parental (p) and Bcr/Abl-expressing (B) Ba/F3 cells, or NIH 3T3 cells (-) are shown at far left in each panel as controls. The positions of P210^BCR/ABL, Lyn, and ERK1/2 are indicated at the arrows.