293T cells were transfected with plasmids encoding the IbAr 10200 M segment or ΔMUCΔGP38-M using FuGENE HD (Promega). Transfected 293T cells incubated at 37°C for 24 or 48 hours, after which cells were collected by low-speed centrifugation and lysed in tris lysis buffer [10 mM tris (pH 7.5), 2.5 mM MgCl2, 100 NaCl, 0.5% Triton X-100, leupeptin (5 μg/μl; Sigma-Aldrich), and 1 mM phenylmethylsulfonyl fluoride]. Twenty microliters of sample were mixed with 4× protein sample buffer [0.125 M tris (pH 8.0), 1% SDS, 0.01% bromophenol blue, and 10% sucrose] with the addition 10× reducing buffer. Protein samples then were analyzed by 10% SDS–polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad Laboratories). PVDF membranes were blocked for 2 hours in PBS [10 mM tris (pH 8.0), 150 mM NaCl, and 0.05% Tween 20] containing 5% nonfat dry milk, rinsed with PBST, and incubated with rabbit polyclonal anti-GN antibody (25) (1:1000), and GC was detected using mAb-11E7 (1:1000). Membranes were subsequently washed with PBST and incubated for 1 hour with horseradish peroxidase–conjugated anti-rabbit (anti-GN; 1:5000 in PBST) or anti-mouse (mAb-11E7) (1:10000 in PBST; Amersham). Bound antibody was detected by treating the PVDF with SuperSignal West Femto chemiluminescent substrate detection reagents (Pierce) and photographed on a G-box (Syngene).

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