Figure 1

Recognition of anaphase-promoting complex (APC)7 antigen with anti-mouse APC7 antibodies. (A) Panel a shows immunoblotting of human MCF-7 breast carcinoma cell extracts with affinity-purified anti-mouse APC7 polyclonal antibodies before (-) and after (+) prebinding APC7 antibodies to recombinant mouse APC7-coupled nitrocellulose. Panel b shows immunoblotting with human APC7 polyclonal antibodies (Santa Cruz; sc-20987). Panel c shows immunoblotting of human Hela cervical carcinoma, mouse NIH 3T3 fibroblast, and human breast carcinomas (MCF, MCF-7; MDA, MDA-MB-231; SK, SK-BR-3; HS, HS 578T) with purified mouse APC7 antibodies. Cell extracts were resolved on 12% SDS-PAGE and the separated proteins were electrotransferred onto Immobilon membranes (Millipore). The membranes were then treated with anti-APC7 antibodies and horseradish peroxidase-conjugated anti-rabbit IgG antibodies as primary and secondary antibodies, respectively. Immune reactive bands were developed using an electrogenerated chemiluminescence (ECL) system. (B) Immunoprecipitation and immunoblotting of mouse 3T3 and human Hela cell extracts with anti-human APC3 (CDC27; Transduction Laboratory) or purified anti-mouse APC7 antibodies. After mixing cytosolic extracts (1 mg) with pre-immune rabbit sera (2 μl), anti-APC3 (1 μg), or anti-mouse APC7 antibodies (1 μg), precipitates were fractionated on 12% SDS-PAGE. After electrotransfer, immunoblotting was performed with anti-APC3, anti-human APC6 (Santa Cruz Biotech), or anti-mouse APC7 antibodies as primary antibodies. Peroxidase-conjugated anti-mouse, anti-goat, or anti-rabbit IgG antibodies were bound as secondary antibodies and developed using the ECL system. (C) Immunohistochemistry of normal breast tissues with purified anti-mouse APC7 antibodies before (panel a) and after (panel b) prebinding APC7 antibodies to recombinant mouse APC7-coupled nitrocellulose (original magnification ×200). After binding the purified anti-mouse APC7 antibodies and the biotinylated goat anti-rabbit antibodies as primary and secondary antibodies, respectively, a streptavidin-peroxidase kit (DAKO) was used to detect APC7 antigen. Staining and counter-staining were then performed using 3-amino-9-ethylcarazole and hematoxylin, respectively.