Pcmv6 ac bap1 plasmid

Human hematopoietic (erythroid/megakaryoblastic) UT-7 parental cell line (UT-7/p) and its counterpart transduced with BCR-ABL-p210 (UT-7/11 cells) were previously described [17] . UT-7/T315I cells, expressing an imatinib-resistant BCR-ABL-T315I mutant, were generated by transfection of 5 mg of a MIGR-BCR-ABL1-T 315I plasmid, which encodes green fluorescent protein (GFP) as reporter, using a lipofection procedure (Life Technologies, Carlsbad, CA). GFP-positive cells were sorted by fluorescenceactivated cell sorting (FACS) and amplified. UT-7/p, UT-7/11, and UT-7/T315I cell lines have been extensively characterized by our group (BCR-ABL protein expression, BCR-ABL activation/phosphorylation, kinase domain mutation screening, and sensitivity toward tyrosine kinase inhibitors) [6, 18] . UT-7/11 cells stably expressing BAP1 were generated by transfection with the pCMV6-AC-BAP1 plasmid (Origene, Rockville, MD) using a lipofection procedure (Life Technologies). Individual G418-resistant clones were expanded in the same culture medium used for UT-7/p but in absence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and in presence of 1 mg/ mL G418. The proteasome inhibitor MG-132 (Sigma-Aldrich, St Louis, MO) was used at 10 mmol/L for 4 hours and imatinib (Novartis, Basel, Switzerland) at 1 mmol/L for 8 or 16 hours.