Fig. 5. ERBB2 kinase domain mutations hyperactivate the PI3K/AKT/m TOR pathway.

(A) Phospho-kinase array of lysates from MCF7/HER2 mutant cells grown in IMEM phenol-red free medium, 5% CSS FBS ± 1 nM estradiol (E₂). Lysates and blots were processed following the manufacturer’s instructions. S6K site T389 shown next to positive control reference (Ref) spots. (B) Immunoblot to confirm array results. The indicated cells were grown in IMEM phenol-red free medium ± 1 nM estradiol overnight. Cell lysates were prepared and subjected to immunoblot analyses with the indicated antibodies. (C) The indicated MCF7 cells were grown in full medium ± 1 μM fulvestrant (F) ± 200 nM neratinib (N). Cell lysates were subjected to immunoblot analyses with the indicated antibodies. (D) Cells were seeded in full medium with 200 nM neratinib, 1 μM alpelisib, 25 nM everolimus, or 1 μM selumetinib, as indicated, ± 1 μM fulvestrant. Cells were seeded in triplicate in 12-well plates and stained on day 10 with crystal violet and imaging intensity of stained monolayers quantitated as described in Methods.