FIGURE 2.
Specific, cytoplasmic association of FLAG-Ago2 with exported (mt) tRNAMet. (A) A 293 cell line was prepared that expresses FLAG-Ago2 after tetracycline induction. Lysates from non-induced (−) and from tetracycline induced (+) 293 cells were probed on a Western blot with anti-FLAG antibody. (B) Cytosolic lysates were prepared from uninduced and from tetracycline-induced cells. The cells were lysed under conditions that preserve the mitochondrial integrity and the lysates were spun to pellet the mitochondria. The post-mitochondrial supernatants (cytosolic lysates) were subjected to immunoprecipitation (IP) with anti-FLAG antibodies. RNA was isolated, resolved on a 10% UREA-PAGE, blotted on a membrane, and probed with radiolabled oligos against the indicated (mt) tRNAs. (Lane 1) Cytosolic IP from uninduced 293 cells; (lane 2) cytosolic IP from tetracycline-induced 293 cells. The blot also contains total RNA isolated from cytosolic lysates (C, lane 3) and from total lysates (T, lane 4) of tetracycline-induced cells. (C) The cytosolic lysate (C) and the total lysate (T), from tetracycline-induced cells, were analyzed by Western blot with an antibody against the mitochondrial COX4 protein. COX4 is not detected in the cytosolic lysate, confirming the absence of mitochondrial contamination.