Lapatinib

TAS0728 was synthesized at the Tsukuba Research Institute, Taiho Pharmaceutical Co., Ltd., by procedures described in our previous study.¹⁴ Trastuzumab, pertuzumab, and T‐DM1 were purchased from Chugai Pharmaceutical Co., Ltd. Lapatinib was purchased from LC Laboratories. For in vivo experiments, TAS0728 was dissolved in 0.1 N HCl solution or in 0.1 N HCl with 0.5% hydroxypropyl methylcellulose (HPMC). Trastuzumab, and T‐DM1 were dissolved in distilled water and diluted with normal saline purchased from the Otsuka Pharmaceutical Factory, Inc. Pertuzumab was diluted with the normal saline. Lapatinib was suspended in a vehicle as described previously.¹⁷The following antibodies were purchased from Cell Signaling Technology Japan, KK and used for western blotting: anti‐phospho‐HER2 (Tyr1196; #6942), anti‐HER2 (#2165), anti‐phospho‐HER3 (Tyr1289; #4791), anti‐HER3 (#12708), anti‐phospho‐AKT (Ser473; #4060), anti‐AKT (#4685), anti‐phospho‐p44/42 MAPK (Thr202/Tyr204; #4370), and anti‐p44/42 MAPK (#4695).

Lapatinib was purchased from Cell Signaling Technology (Berverly, MA), and cisplatin (CDDP) was purchased from Sigma (St. Louis, MO). Antibodies against phospho-HER2^Tyr1221/1222 (pHER2, rabbit monoclonal), HER2 (rabbit monoclonal), phospho-MET^Tyr1234/1235 (pMET, rabbit monoclonal), phospho-AKT^Ser473 (pAKT, rabbit polyclonal), AKT (rabbit polyclonal), and FOXO1 (rabbit monoclonal) were purchased from Cell Signaling Technology. Antibodies against MET (rabbit polyclonal), β-actin (mouse monoclonal) and secondary antibodies, which are horseradish peroxidase-conjugated anti-rabbit IgG or anti-mouse IgG, were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).