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PFigure 4 Site-directed mutagenesis analysis of the three predicted AREs. (A) The wild-type GNMT promoter reporter construct was generated by cloning a 1.2 kb region of the GNMT gene into a pGL3-basic luciferase vector. .l-]  6 X  x @`j  BA ?1mj  BA ?TA p  T$3z ?3s  pSilvia Ottaviani et al. J Mol Endocrinol 2013;51:301-312,9l-]9  9 X J  T3z ?D Z @ 2013 Society for Endocrinology*!l-]!  ! & X  x 6N  6qљYs ? ̙33ZR___PPT102.]0G+D' = @B D' = @BA?%,( < +O%,( < ++  5   @0 (  @  C ?z7   T3z ? 92   Site-directed mutagenesis analysis of the three predicted AREs. (A) The wild-type GNMT promoter reporter construct was generated by cloning a 1.2 kb region of the GNMT gene into a pGL3-basic luciferase vector. This construct was used as a template for site-directed mutagenesis of ARE-I (ARE-I*), ARE-II (ARE-II*) and ARE-III (ARE-III*). The mutants were generated by replacing part of the ARE sequence with the MluI restriction site sequence (52 -acggct-32 ). (B) The activity of the GNMT ARE mutants was tested using the luciferase reporter assay. Firefly luciferase activities were normalised for transfection efficiency against the Renilla luciferase activities. Results are shown as mean of three independent experiments, each performed in triplicate. Error bars represent the s.e.m. Statistical significance was calculated by unpaired two-tailed Student's t-test: **P<0.01.n0 l-]R# # M# # <# # # # # # # # #  n & X  x 6H  0޽h ? ̙33rP> ^t p)Root EntrydO)CompObj9Ole Current User,Pictures[SummaryInformation(xPowerPoint Document(J)DocumentSummaryInformation8