FRAT proteins mediate MLL fusion-induced Rac activity. (A) Expression of Frat1, Frat2, and Dvl1 mRNA in preleukemic MLL-ENL cells treated with and without doxycycline for 72 hours, as determined by quantitative PCR. Bars represent means of quadruplicate values; and error bars, the SD. Data are representative of 3 independent experiments. (B) Quantitative PCR analysis of Frat1 and Frat2 mRNA expression in leukemic MLL-ENL cells transduced with Scrambled (Sc), Frat1 (F1a and F1b), and Frat2 (F2a and F2b) shRNA retroviral vectors. Bars represent means of quadruplicate values; and error bars, the SD. Data are representative of 3 independent experiments. (C-D) Western blot analysis of Rac1 GTP and Rac2 GTP levels, and total Rac protein expression (C) in leukemic MLL-ENL cells transduced with Scrambled (Sc), Frat1 (F1a and F1b), and Frat2 (F2a and F2b) shRNA retroviral vectors, and (D) in preleukemic MLL-ENL cells transduced with control (Con), Frat1 (F1), and Frat2 (F2) cDNA expressing retroviral vectors. Numbers represent densitometric quantitation of Rac GTP levels normalized to total Rac protein bands. α-tubulin served as a loading control. Data are representative of 2 independent experiments. (E) Number of colonies formed in methylcellulose by leukemic MLL-ENL cells transduced with Scrambled (Sc) and Frat2 (F2a and F2b) shRNA retroviral vectors. Bars represent means of duplicate values; and error bars, the SD. **P < .01 versus control. Data are representative of 3 independent experiments. (B-C,E) At 48 hours after shRNA transduction, cells were selected with puromycin for 72 hours and then harvested for analysis or plated into methylcellulose. (D) At 48 hours after transduction, hCD2T⁺ transduced cells were positively selected by MACS and then harvested for protein lysates 5 days later.