Fig. 3. Extended inhibition of GSK3ß causes a robust reduction of APC in the 23S complex. (A,B) HeLa cells were treated with the GSK3ß inhibitors, SB216763 (5 µM) (A) or LiCl (20 mM) (B) for 12 hours. Cell lysates were fractionated and the distribution of APC and ß-catenin determined as above. Inhibiting GSK3ß for 12 hours greatly reduced the amount of APC in the 23S complex and also resulted in a higher proportion of ß-catenin cofractionating with APC in the 10S complex. This effect was produced more effectively by SB216763, the more specific of the two inhibitors. These plots represent the distribution profiles of two independent experiments. (C) Immunoblots of equal amounts of total cell lysates were probed with antibodies against APC and ß-catenin. This confirmed that both GSK3ß inhibitors produced an increase in the cellular ß-catenin content, whereas the amount of APC remained constant. (D) APC was immunoprecipitated from cell lysates of SB216763 and LiCl treated HeLa cells as indicated. Probing the immunoprecipitated material with antibodies against ß-catenin revealed an increased amount of ß-catenin bound to APC when GSK3ß was inhibited. (E) Total cell lysates were also probed with an antibody against the phospho-epitope Y-216 of GSK3 or a GSK3ß-specific antibody (lower panel). Only SB216763 produced a decrease in the activating phosphorylation of GSK3 and GSK3ß at Y-216, consistent with the fact that LiCl and SB216763 inhibit GSK3 by different mechanisms.