Epr20043

Cells were collected following a brief exposure to 0.25% trypsin and 1 mM ethylenediaminetetraacetic acid (EDTA; Nacalai Tesque, Inc.). The cells were washed with 0.1% bovine serum albumin in phosphate-buffered saline (PBS) and treated with anti-TROP2 mAbs, such as TrMab-6 (1 µg/ml) or EPR20043 (1/60 dilution; Abcam) for 30 min at 4°C. After incubation, the cells were treated with Alexa Fluor 488-conjugated anti-mouse IgG (1:1,000; Cell Signaling Technology, Inc.) or Alexa Fluor 488-conjugated anti-rabbit IgG (1:1,000; Cell Signaling Technology, Inc.). Fluorescence data were collected using SA3800 Spectral Cell Analyzer (Sony Corp.) and analyzed using FlowJo (BD Biosciences).

Paraffin-embedded tissue sections of the breast cancer tissue array (Cat#T8235721-5, Lot#B104066; BioChain, San Francisco, CA, USA) were autoclaved in EnVision FLEX Target Retrieval Solution High pH (Agilent Technologies, Inc.) for 20 min. After blocking with SuperBlock T20 (Thermo Fisher Scientific, Inc.), tissue sections were incubated with TrMab-6 (5 µg/ml) or EPR20043 (1/500 dilution; Abcam) for 1 h at room temperature and then treated with the EnVision+ Kit for mouse (Agilent Technologies Inc.) and EnVision+ Kit for rabbit (Agilent Technologies Inc.) for 30 min, respectively. Color was developed using 3,3′-diaminobenzidine tetrahydrochloride (DAB; Agilent Technologies Inc.) for 2 min. Counterstaining was performed with hematoxylin (FUJIFILM Wako Pure Chemical Corporation).

Cell lysates (10 µg) were boiled in sodium dodecyl sulfate (SDS) sample buffer (Nacalai Tesque, Inc.). Proteins were separated on 5–20% polyacrylamide gels (FUJIFILM Wako Pure Chemical Corporation) and transferred onto polyvinylidene difluoride (PVDF) membranes (Merck KGaA). After blocking with 4% skim milk (Nacalai Tesque, Inc.) in PBS with 0.05% Tween-20, the membranes were incubated with 1 or 5 µg/ml of TrMab-6, 1/2000 dilution of EPR20043 (Abcam), 1 µg/ml of NZ-1 (anti-PA tag), or 1 µg/ml of anti-β-actin (clone AC-15; Sigma-Aldrich Corp.). This was followed by incubation with peroxidase-conjugated anti-mouse immunoglobulins (Agilent Technologies Inc.; diluted 1:1,000) to detect TrMab-6 and anti-β-actin, peroxidase-conjugated anti-rabbit immunoglobulins (Agilent Technologies Inc.; diluted 1:1,000) to detect EPR20043, or anti-rat IgG (Sigma-Aldrich Corp; diluted 1:10,000) to detect NZ-1, respectively. Finally, protein bands were detected with ImmunoStar LD (FUJIFILM Wako Pure Chemical Corporation) using a Sayaca-Imager (DRC Co. Ltd.).