PMC

Full-length APC protein locates to both membrane/cytoskeletal and nuclear cell fractions in LS174T cells. Proteins within the various fractions of LS174T cells were analyzed by SDS/PAGE and immunoblotting. Fractions are labeled at the figure bottom as follows: T, total; C, cytoplasm; M, membrane/cytoskeleton; N, nucleus; Sc, nuclear scaffold. The antibodies used for the Western immunoblots are as follows: a, APC;, b, tubulin as a cytoskeletal marker; c, β-adaptin as a membrane marker; and d, lamins as nuclear and nuclear matrix scaffold markers.

Nuclear APC protein co-localizes with rRNA. 184A1 cells were stained simultaneously for APC protein (a) and rRNA (b) using antibodies specific for the APC protein and a fluorescein isothiocyanate-conjugated DNA oligo complementary to rRNA. The yellow staining in c reflects the colocalization of rRNA and APC protein. Solid arrowheads designate nuclear staining in a single cell (a–c). Open arrowheads designate additional regions of staining overlap. d is a DIC and DAPI view of the fluorescence views shown in a–c. (Bar = 10 μm.)

Truncated APC protein does not fractionate with the nucleus of DLD-1 cells. Proteins within the various fractions of DLD-1 cells were analyzed by SDS/PAGE and immunoblotting. Fractions are labeled as follows: T, total; C, cytoplasm; M, membrane/cytoskeleton; N, nucleus; Sc, nuclear scaffold. The antibodies used for the Western immunoblots include: a, APC; b, tubulin as a cytoskeletal marker; c, β-adaptin as a membrane marker; and d, lamins as nuclear and nuclear matrix scaffold markers. Truncated APC protein is present in the membrane/cytoskeletal fraction, but not the nuclear or nuclear scaffold fractions.

Antibodies recognize different epitopes of the APC protein. Schematic representation of the APC protein as adapted from (). The mutation cluster region is indicated by the oval. β-catenin-binding region is indicated by a shaded rectangle. □, Oligomerization region; ○, mAbs that recognize distinct APC epitopes used in the present study. Antibody Ab-4, whose epitope has not been mapped precisely, was made against a 300-amino acid peptide beginning at • and proceeding to the end of the APC protein. The solid line represents the APC region that was used to produce the polyclonal rabbit sera APC64. The arrow marks the point of APC protein truncation in the colon cancer cell line DLD-1. The circled Ns·· indicate two potential nuclear localization sequences beginning at amino acids 1773 and 2054.

Localization of APC protein in 184A1 cells using immunofluorescence microscopy. 184A1 cells were grown on glass slides prior to fixation and immunofluorescence microscopy using Ab-4, an antibody specific for APC protein (b and c) or using Ab-4 preincubated with an APC peptide (e). b and c are photographs of the same group of cells taken at two focal distances to more clearly capture cell edge staining (b, solid arrowhead) and nuclear staining (c, open arrowhead). APC protein appears in a punctate pattern throughout the cytoplasm with areas of protein concentration at one edge (solid arrowhead). In addition, APC protein appears throughout the nuclei with a few areas of concentration (c). a and d are DIC and DAPI views of the fluorescence views shown in b, c, and e, respectively. Controls include: f, 184A1 cells stained with nonspecific antibody IgG₁; g–i, 184A1 cells stained for APC using antibodies Ab-2 (g), Ab-6 (h) or APC64 (i); j, APC staining of T47D cells. For each antibody, both edge staining (solid arrowhead) and nuclear staining (open arrowhead) are apparent. In k, DLD-1 cells that express only truncated APC protein were stained using the C terminal antibody Ab-4 to demonstrate staining specificity. l is the corresponding DIC and DAPI view of the cells shown in k. (Bar = 10 μm.)