32D cells expressing mutated PDGFRA retain ligand dependent phosphorylation of AKT and imatinib sensitivity in vitro. (A) Activation of different signaling pathways by PDGF-AA. Cell mutants were starved overnight in medium containing 0.5% FCS and stimulated with 10 ng/mL PDGF-AA for 10 minutes. Total cell lysates were prepared, and the Western blot membranes were stained with the indicated phosphorylation-specific antibodies. (B) MTS assay in presence of imatinib and calculated IC50 values. The same 32D cells were used in a Cell Titer 96 Aqueous Solution Assay to measure the influence of imatinib on proliferation after 72 hours of imatinib exposure. Each point represents the mean percentage of growth compared with the untreated cells from 3 independent experiments. Data were used to calculate IC50 values with GraphPad Prism 5. (C) Inhibition of intracellular signaling by imatinib treatment. 32D cell mutants were starved overnight in medium containing 0.5% FCS and treated with the indicated concentrations of imatinib for 2 hours. Total cell lysates were separated by SDS-PAGE. After blotting, the blots were stained with a phosphorylation-specific Stat5, total Stat5, or β-actin antibody. (D) FACS analysis of imatinib-treated mutant 32D cell lines. Cells were incubated with 0.5 or 5μM imatinib or vehicle for 72h and 7-AAD stained. Depicted percentages and gates represent remaining GFP-positive and 7-AAD negative cells. The figure shows 1 representative experiment of 2 independent experiments.