The BCR-ABL^35INS mutant does not confer growth factor independence or imatinib resistance, and ABL^35INS lacks kinase activity. (A) Ba/F3 cells expressing the indicated BCR-ABL construct were cultured in standard media supplemented with IL-3. After IL-3 washout and replating in standard media, cell viability was monitored daily for 4 days with the Guava ViaCount assay (Millipore). Results are shown as the mean viable cells ± SEM from 3 independent experiments. (B) Cell lysates were subjected to SDS-PAGE followed by immunoblot (IB) analysis with the indicated antibodies. Results are representative of 2 independent experiments. The long and short arrows on the right side of the immunoblot indicate the expected positions of BCR-ABL and BCR-ABL^35INS, respectively. (C) Ba/F3 cells stably expressing BCR-ABL were transduced with the indicated BCR-ABL^35INS mutant constructs, sorted for GFP, and cultured in standard media. Cells were plated in the presence of graded concentrations of imatinib (0-3125nM) and evaluated by methanethiosulfonate assay at 72 hours (CellTiter 96 AQ_ueous One Solution Cell Proliferation Assay; Promega) with a Synergy 2 plate reader (BioTek). Results are normalized to those of untreated cells and expressed as the mean ± SEM of 3 independent experiments performed in quadruplicate. (D) Cell lysates were subjected to SDS-PAGE followed by immunoblot (IB) analysis with the indicated antibodies. The long and short arrows on the right side of the immunoblot are provided for reference and indicate the expected positions of BCR-ABL and BCR-ABL^35INS, respectively. The absence of signal corresponding to BCR-ABL^35INS in the second and third panels signifies the lack of the ABL C-terminus and no phosphorylation on Y393, respectively. Lanes 1 and 2 represent nonadjacent lanes from the same gel (indicated by the vertical dashed line). (E) Equimolar amounts of purified, tyrosine-dephosphorylated GST-ABL kinase domain enzymes were incubated with [γ-³²P]-ATP. Samples collected at indicated times were subjected to SDS-PAGE, and signal intensity was measured by autoradiography. Comparable loading was confirmed by ABL immunoblot (IB) analysis with ABL antibody Ab-2 (Oncogene Science). Results are representative of 2 independent experiments. (F) Dephosphorylated GST-ABL enzymes were incubated with a biotinylated peptide substrate (biotin-GGEAIYAAPFKK-amide) and [γ-³²P]-ATP. At the end of the incubation period, reactions were terminated with 7M of guanidine hydrochloride solution, spotted in duplicate on SAM² Biotin Capture Membranes (Promega), washed according to the manufacturer's instructions, and subjected to liquid scintillation counting. GST-ABL reactions were quenched after 3.5 minutes, whereas GST-ABL^35INS and GST-ABL^K271R/35INS reactions were quenched after 1 hour because preliminary experiments at 3.5 minutes demonstrated no detectable activity. Results are shown as the mean enzymatic activity ± SEM normalized to that of GST-ABL. The autophosphorylation and peptide substrate phosphorylation activities of GST-ABL were inhibited by imatinib (data not shown). WT indicates wild-type.