Fig. 2. Axin-1 co-sediments with the 23S APC-containing complex and is re-distributed to the 10S complex after cells were treated with GSK3ß inhibitor. Cell lysates were fractionated on 10-40% glycerol gradients as in Fig. 1. (A) Every other fraction was subjected to PAGE and probed with antibodies against APC, ß-catenin, GSK3ß, and Axin-1. (B) Intensities of the bands were measured using a CCD-based ECL detection system and then plotted as indicated. The majority of cellular Axin-1 co-sedimented with the 23S APC-containing complex. (C) Parallel samples of cells were treated with the GSK3ß inhibitor SB216763 (20 µM) for 2 hours and processed as described for A. Treatment of cells with SB216763 resulted in a relative increase in the proportion of APC and Axin-1 in smaller complexes. (D) Cell lysates from HeLa cells treated with SB for 12 hours were fractionated and probed for APC, Axin-1 and GSK3ß. Axin-1 distributes almost exclusive to the 10S complex after 12 hours of GSK3ß inhibition, similar to APC. (E) Cell lysates of cells treated with SB216763 for 2 hours were probed with antibodies against APC, ß-catenin, GSK3ß and tubulin. Only ß-catenin was increased whereas the levels of the other proteins remained constant. (F) Cell lysates from control or SB216763-treated cells were immunoprecipitated with a monoclonal antibody against APC. Immunoprecipitated material was probed with antibodies against APC and ß-catenin. Despite the elevated levels of ß-catenin in SB216763-treated cells, there was no concomitant increase in the amount of ß-catenin bound to APC.