Figure 4.

Ezrin, moesin, and phosphorylated merlin are complexed with CD44 at low cell density. Ezrin, moesin, and doxycycline-induced (A) or endogenous (B) phosphorylated merlin are coprecipitated with CD44. For coimmunoprecipitation experiments, clone 6₇ cells and parental RT4-D6P2T were seeded at low cell density. All procedures were as in Fig. 3C and D. Lysates of clone 6₇ cells at high cell density (conf) were included to control for the resolution of merlin modifications. Overexpressed wild-type cytoplasmic tail of CD44 pulls down merlin, ezrin, and moesin from lysates of cells at low cell density (C). Cells of clone 6₇ transfected with either wild-type or mutant CD44 tail constructs, were plated at low cell density. Eight hours after doxycycline addition, the cells were lysed and the CD44 tails enriched by GSH agarose. Procedures as in Fig. 3F. Phosphorylated merlin coprecipitates with ezrin and moesin at low cell density (D). Clone 6₇ cells were plated at low or high density and treated with doxycycline for 8 h. Coimmunoprecipitation was performed as described in A, except for the use of an antiezrin/moesin antibody (C-19) for the precipitation. Lysates were subjected to a higher resolution gel. Western blotting as in A.