Lapatinib-resistant PIK3CA mutant cell lines show reactivation of phosphoinositide 3-kinase and utilize mutant p110α for phosphoinositide 3-kinase signaling. (A) Cells expressing E545K or H1047R were selected for lapatinib resistance. Unselected cells (Pre) were treated with lapatinib for 3 hours and analyzed along with lapatinib-resistant (LR) cells in the presence of lapatinib by immunoblotting with the indicated antibodies. (B) Lapatinib-sensitive and LR cells were treated with lapatinib and/or BKM120 for 3 hours, and lysates were analyzed by immunoblotting. (C) Phosphoinositide 3-kinase (PI3K) was immunoprecipitated from PIK3CA mutant–expressing cells before and after selection for lapatinib resistance with a p85 antibody. Complexes were separated by SDS-PAGE, and expression of mutant p110α was determined by immunoblot analysis for the hemagglutinin (HA)-tag. (D) HA band intensity detected by infrared fluorescence was quantified using a LI-COR Odyssey imaging system. Mean HA intensity normalized to p85 intensity from at least four replicate experiments is shown (bars = SEM).