FIGURE 8.

Dimerization is required for nuclear export of TonEBP. A, FLAG-tagged TonEBP or its site-directed mutant, DIM (F464A/I466A) or DBM (T298A/E299A/R302A), was coexpressed with Myc-TonEBP in COS7 cells. Cell lysates were immunoprecipitated (IP) with anti-FLAG antibody. The immunoprecipitates, and cell lysates were immunoblotted (IB) for FLAG or Myc, as indicated. B, the FLAG-tagged constructs were transfected into COS7 cells. Top, cell lysates were immunoblotted for FLAG. Bottom, EMSA was performed using the cell lysates and 1 nm [³²P]TonE. The arrow denotes the TonEBP·DNA complex. C, COS7 cells transfected as in B were seeded on coverslips and analyzed as in Fig. 3 using FLAG immunofluorescence. Fluorescence images of the cells in hypotonic conditions are shown at the bottom.