Hrp conjugated anti mouse immunoglobulins

Elution fractions and cell lysates were boiled in SDS sample buffer (Nacalai Tesque, Inc.). The samples were electrophoresed on 5%–20% polyacrylamide gels (Nacalai Tesque, Inc.), and were transferred onto polyvinylidene difluoride (PVDF) membranes (Merck KGaA, Darmstadt, Germany). After blocking with 4% skim milk (Nacalai Tesque, Inc.) for 1 h, the membranes were incubated with LpMab-7 (1 μg/ml) or PMab-237 (1 μg/ml) for 1 h, followed by HRP-conjugated anti-mouse immunoglobulins (1:1000 dilution; Agilent Technologies, Inc., Santa Clara, CA, USA) for 30 min. The membrane was also incubated with anti-CD20 mAb (EP459Y; 1:1000 dilution; Abcam, Cambridge, UK) for 1 h, followed by anti-rabbit immunoglobulins (1:1000 dilution; Agilent Technologies, Inc.) for 30 min. The membranes were visualized with the ImmunoStar LD Chemiluminescence Reagent (FUJIFILM Wako Pure Chemical Corporation) using the Sayaca-Imager. All procedures of Western blotting were performed at room temperature.

Lysates were boiled in sodium dodecyl sulfate sample buffer (Nacalai Tesque, Inc., Kyoto, Japan). The samples were electrophoresed using 5%–20% polyacrylamide gels under reducing condition (Nacalai Tesque, Inc.), and transferred onto a polyvinylidene difluoride (PVDF) membranes (Merck KGaA, Darmstadt, Germany). After blocking with 4% skim milk (Nacalai Tesque, Inc.) for 1 h, the membrane was incubated with DdMab-1 (1 μg/mL or 10 μg/mL) or NZ-1 (1 μg/mL) for 1 h, followed by incubation with HRP-conjugated anti-mouse immunoglobulins (1:2000 dilution; Agilent Technologies, Inc.) or HRP-conjugated anti-rat IgG (1:10,000 dilution; Sigma-Aldrich Corp.) for 1 h. The membrane was developed with the ImmunoStar LD Chemiluminescence Reagent (FUJIFILM Wako Pure Chemical Corporation) using the Sayaca-Imager (DRC Co., Ltd., Tokyo, Japan). All Western blot procedures were performed at room temperature.

Cell lysates of CHO/EpCAM, CHO-K1, Caco-2, and BINDS-16 cells were boiled in sodium dodecyl sulfate sample buffer (Nacalai Tesque, Inc.). The samples were then electrophoresed on 5–20% polyacrylamide gels (Nacalai Tesque, Inc.) and transferred onto polyvinylidene difluoride (PVDF) membranes (Merck KGaA, Darmstadt, Germany). After blocking with 4% skim milk (Nacalai Tesque, Inc.) for 1 h, the membrane was incubated with recEpMab-37 (1 μg/mL) or anti-β-actin (1 μg/mL) for 1 h, followed by incubation with HRP-conjugated anti-mouse immunoglobulins (Agilent Technologies, Inc., Santa Clara, CA, USA) at a 1:2000 dilution for 1 h. The membrane was developed using the ImmunoStar LD Chemiluminescence Reagent (FUJIFILM Wako Pure Chemical Corporation) and a Sayaca-Imager (DRC Co., Ltd., Tokyo, Japan). All Western blot procedures were performed at room temperature.