Fig. 7. Prolonged overexpression of Dsh induces expression of DE-cadherin. (A) Untransfected cl-8 cells and cl-8 cells expressing Dsh under control of the metallothioneine promoter (cl8Dshmyc) (Yanagawa et al., 1995) were incubated for various times with Cu²⁺, and whole cell lysates at each time point were analyzed by western blotting with the indicated antibodies. Both the amounts of overexpressed Dsh and of hypophosphorylated Arm reached steady state levels after 4 hours of induction with Cu²⁺. At 12 hours of induction, DE-cadherin levels began to increase, which correlated with the appearance of a slower migrating, phosphorylated form of Arm. Levels of -catenin remained unaffected by Dsh overexpression. Untransfected cl-8 cells did not show a significant change in any of the proteins analyzed after incubation with Cu²⁺. (B) Northern blot analysis of total RNA of cl-8 cells and cl8Dshmyc cells incubated with Cu²⁺ for various times. In cl8Dshmyc cells, levels of DE-cadherin mRNA were constant during the first 12 hours of Dsh induction and increased strongly between 12 and 24 hours. Levels of Arm mRNA did not change significantly between 0 and 24 hours. Incubation with Cu²⁺ had no effect on DE-cadherin or Arm mRNA levels in untransfected cl-8 cells.