Figure 4.

Western blots with the anti-FLAG antibody for the detection of amber suppression in CHO cells. (A) The ras gene (lane 1) or the ras(Am) gene (lanes 2–8), each with the FLAG tag, was introduced into the cells. The human suppressor tRNA^Tyr (lane 3) or the pair E.coli TyrRS and E.coli suppressor tRNA^Tyr (lane 4) was expressed in the cells. This enzyme also has the FLAG tag. The B.stearothermophilus suppressor tRNA^Tyr was expressed, together with the E.coli TyrRS (lanes 5 and 8) or without the enzyme (lane 7), or the E.coli TyrRS alone was expressed in the cells (lane 6). The B.stearothermophilus suppressor tRNA^Tyr was expressed from the plasmid carrying a single copy of its gene (lanes 5 and 7) and from that carrying nine copies (lane 8). A 2.5 µg aliquot of cell extract was subjected to SDS–PAGE in lane 1, while a 4-fold greater quantity of the cell extract was analyzed in lanes 2–8. The protein detected in all of the cell extracts (lanes 1–8) is an endogenous protein in CHO cells. (B) The epidermal growth factor receptor (EGFR) gene containing an amber codon in position 1068 (lanes 1 and 3) and the wild-type EGFR gene (lane 2), each with a FLAG tag, were introduced into CHO cells. The B.stearothermophilus suppressor tRNA^Tyr and the E.coli TyrRS pair were expressed in the cells (lane 3). The faint band migrating at the level of the E.coli TyrRS (lanes 1 and 2) was due to endogenous proteins that respond to the anti-FLAG antibody.