Specific binding of pertuzumab to HER2. (A) The expression levels of HER2 in CHO cell lines stably overexpressing HER2 (CHO-K13 and CHO-K6), two HER2-positive (SKBR-3 and BT-474) and two HER2-negative (MCF-7 and MDA-MB-231) breast cancer cell lines. (B) Double-immunofluorescence staining of EGFR, HER2, and HER3 (green) and pertuzumab (P; red) in CHO-EGFR, CHO-K6, and CHO-HER3 cell lines. Pertuzumab binds specifically to HER2 but not to EGFR and HER3. (C,D) Double-immunofluorescence staining of HER2 (green) and pertuzumab (red) in the cells (C) treated with 0.1, 0.5, 1, 5, and 10 μg/mL pertuzumab for 60 min (dose-course treatment) and (D) treated with 10 μg/mL pertuzumab for 5, 15, 30, and 60 min (time-course treatment). Treatment with 10 μg/mL of human IgG for 60 min was used as a mock control.