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Fig. 1. Fractionation of APC, ß-catenin and GSK3ß from different cell lines on glycerol gradients. Cell lysates from the indicated cells were fractionated on 10-40% glycerol gradients. Every other fraction was subjected to PAGE, transferred to nitrocellulose probed with antibodies against APC, ß-catenin and GSK3ß. The relative intensity of each protein was determined using a charge-coupled-device (CCD)-based enhanced chemiluminescence (ECL) detection system and was plotted for each fraction. Sedimentation of proteins with known S-values was carried out in parallel for each experiment to allow calibration for each gradient. In HeLa and MDCK cells, which express the intact ß-catenin-targeting complex, APC was present in two pools that sedimented at 10S and 23S. However, in cells that only express truncated APC or mutated ß-catenin, which cannot be phosphorylated, the relative amount of APC in the 23S complex was reduced or absent (SW480, DLD-1, HCT 116) or was shifted to a smaller size complex (HT-29).
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