Western Blot Analysis of C44 Antibodies

The SDS-polyacrylamide gel for electrophoresis and transfer onto polyvinylidene difluoride membranes was achieved as described previously [28 (link)]. After the blocking in PBST containing 4% skim milk (Nacalai Tesque, Inc.), the membranes were incubated with 10 μg/mL of C₄₄Mab-46, 10 μg/mL of C₄₄Mab-18, or 0.5 μg/mL of an anti-β-actin mAb (AC-15; Sigma-Aldrich Corp.). The membranes were further treated with peroxidase-conjugated anti-mouse immunoglobulins (diluted 1:1000; Agilent Technologies, Inc.). Finally, the chemiluminescence signal was obtained using ImmunoStar LD (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) and was detected using a Sayaca-Imager (DRC Co., Ltd., Tokyo, Japan).