Anti mouse immunoglobulins conjugated with peroxidase

The cell lysates (10 μg of protein) were prepared using sodium dodecyl sulfate (SDS) sample buffer (Nacalai Tesque, Inc.), and separated on 5–20% polyacrylamide gels (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan). The proteins were transferred onto polyvinylidene difluoride (PVDF) membranes (Merck KGaA, Darmstadt, Germany). The membranes were incubated with 10 μg/mL of C₄₄Mab-108, 1 µg/mL of an anti-PA16 tag mAb (NZ-1), or 1 μg/mL of anti-β-actin mAb (clone AC-15; Sigma-Aldrich Corp.) in 4% skim milk (Nacalai Tesque, Inc.) in PBS with 0.05% Tween 20. Then, the membranes were incubated with anti-mouse immunoglobulins conjugated with peroxidase (diluted 1:1000; Agilent Technologies, Inc.) for C₄₄Mab-108 and anti-β-actin. The anti-rat immunoglobulins (diluted 1:10,000; Sigma-Aldrich Corp.) conjugated with peroxidase were used for NZ-1. Finally, the signals were detected with a chemiluminescence reagent, ImmunoStar LD (FUJIFILM Wako Pure Chemical Corporation) using a Sayaca-Imager (DRC Co. Ltd., Tokyo, Japan).

Four peptides, covering the v7, v8, and v9 regions of CD44v3–10, were obtained from Sigma-Aldrich Corp. (St. Louis, MO, USA).
The peptide sequences were as follows.
CD44p421–440 (GHQAGRRMDMDSSHSTTLQP); v7/v8,
CD44p431–450 (DSSHSTTLQPTANPNTGLVE); v8,
CD44p441–460 (TANPNTGLVEDLDRTGPLSM); v8,
CD44p451–470 (DLDRTGPLSMTTQQSNSQSF); v8/v9.
The peptides (10 µg/mL) were immobilized on 96-well immunoplates (Nunc Maxisorp; Thermo Fisher Scientific Inc.). The immunoplate washing was performed with PBS containing 0.05% (v/v) Tween 20 (PBST; Nacalai Tesque, Inc.). The blocking was performed with 1% (w/v) bovine serum albumin (BSA) in PBST. C₄₄Mab-94 (10 µg/mL) or blocking buffer was added to the peptide-coated wells. Next, the wells were further incubated with anti-mouse immunoglobulins conjugated with peroxidase (1:2000 dilution; Agilent Technologies Inc., Santa Clara, CA, USA). The ELISA POD Substrate TMB Kit (Nacalai Tesque, Inc.) was used for the peroxidase reaction. Using an iMark microplate reader (Bio-Rad Laboratories, Inc., Berkeley, CA, USA), the optical density (655 nm) was measured.

The cell lysates (10 μg) were treated with sodium dodecyl sulfate (SDS) sample buffer (Nacalai) for 5 min at 95 °C. The proteins were electrophoresed using 5–20% polyacrylamide precast gels for electrophoresis (Wako) and transferred to polyvinylidene difluoride (PVDF) membranes (Merck KGaA, Darmstadt, Germany). The membranes were blocked using 4% skim milk (Nacalai) in PBS with 0.05% Tween 20 and were treated with 1 μg/mL of H₂Mab-139-mG_2a-f or 1 μg/mL of an anti-isocitrate dehydrogenase 1 (IDH1) mAb (RcMab-1). The membranes were then incubated with anti-mouse immunoglobulins conjugated with peroxidase [diluted 1:1000; Agilent Technologies, Inc. (Agilent), Santa Clara, CA, USA, for H₂Mab-139-mG_2a-f], or with anti-rat immunoglobulins conjugated with peroxidase (diluted 1:1000; Agilent, for RcMab-1). Finally, the signals were detected using a chemiluminescence reagent, ImmunoStar LD (Wako) using a Sayaca-Imager (DRC Co., Ltd., Tokyo, Japan).