Figure 2

APC-NT mAbs specifically detect APC at the ends of microtubules. A) MDCK cells transfected with control siRNA (top) or APC siRNA (bottom) were immunostained for APC (APC-NT mAb, 6D12 (red)) and β-tubulin (green). Distinct clusters of APC at the ends of microtubules (arrowheads) are detected by the APC-NT mAb in control but not APC depleted cells, scale bar 20 μm. Enlarged view of insets is shown (right, merged image (left) and single channel grayscale for APC (right)). Shown are single section confocal images, scale bar 5 μm. B) Depletion of cellular APC by siRNA. APC levels following siRNA transfection were assessed by immunoblot analysis (with H290 Ab from Santa Cruz) in cell lysates. Tubulin was used as a loading control. Numbers below blot are the normalised ratio of APC protein compared to control siRNA. The relative positions of molecular weight markers are indicated. C) SW480 CRC cells were immunostained for APC (APC-NT) and costained with DAPI. APC staining is diffusely cytosolic and is not detected in clusters at the ends of microtubules. Shown are single channel grayscale image for APC (left) and merged image (APC, red and DAPI blue), scale bar 20 μm.