FIG. 6.
Marked box domain of E2F3 is both necessary and sufficient for binding to TFE3. (A) Schematic of the chimeric E2Fs described in this study. The nomenclature describes the identity of individual domains in the chimeras: first digit, amino terminus/cyclin A binding; second digit, DNA binding domain; third digit, DP1 dimerization domain; fourth digit, marked box; fifth digit, marked box adjacent region; sixth digit, transactivation/Rb binding domain. The resultant chimeric E2F cDNAs were cloned into a cytomegalovirus-HA expression vector. (B) NIH 3T3 cells were transfected with Myc-TFE3 and HA-tagged E2F1 to E2F3 chimeric proteins. Transfected cells were lysed in immunoprecipitation lysis buffer 24 h posttransfection. HA antibody immobilized on Sepharose beads (Covance) was used to immunoprecipitate the HA-tagged E2Fs. Myc-TFE3 that coimmunoprecipitated with the E2F proteins was detected by Western blotting with specific antibodies (Co-IP TFE3). The level of TFE3 expressed in the transfected cells was measured by Western blotting of aliquots before the immunoprecipitation (input TFE3). Finally, the amounts of the E2Fs that were recovered in the immunoprecipitations (IP-E2Fs) were determined by Western blotting with HA antibody. NS, nonspecific. (C) NIH 3T3 cells were transfected with the p68 promoter and 10 ng of either empty vector, expression plasmids for E2F1 or E2F3, or the E2F1 to E2F3 chimeric proteins alone or in combination with 100 ng of the TFE3 expression plasmid. TFE3 alone was used as a control. Cells were then harvested for determination of Renilla luciferase activity.