FIG. 6.

Overexpression of LysRS via elevated amounts of Ap₄A increases the transcriptional activity of USF2 and MITF. (A) Ap₄A levels were determined with Lys-101 CHO, CKRS, and CKRS N̂ cells. The measurement of Ap₄A was determined luminometrically as described in Materials and Methods. The bars show means ± standard error for three experiments. In addition, LysRS protein levels in these cell lines are shown at the bottom of the graph. From each cell line, 30 μg of total protein was analyzed by Western blotting with anti-LysRS antibody. Duplicates are shown for each cell line. (B) USF2, MITF, c-Myc, and STAT3 transcriptional activity in Lys-101 CHO cells overexpressing LysRS. CKRS and CKRS^N cells were transfected with a luciferase reporter gene containing the USF2-, MITF-, and c-Myc-responsive elements and a PKA-Cβ reporter gene with STAT3-C. Normalized values are relative severalfold increases in luciferase activity in CKRS cells compared to those in CKRS^N cells. Values are means ± standard error for three experiments. (C) Coimmunoprecipitation of either USF2 or MITF with Hint in cells overexpressing LysRS. CKRS and CKRS^N cells were lysed and incubated with either anti-USF2 or anti-MITF antibody. The resolved immunocomplexes were analyzed by Western blotting with anti-Hint antibody. One representative result of three is shown. (D) The lysates of CKRS and CKRS^N cells were subjected to Western blotting with anti-LysRS, anti-Hint, anti-MITF, and anti-USF2 to show the protein levels in various CHO cells. One representative result of three is shown.