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- _5 value "To investigate whether IGF-1 also enhanced transactivation potential, a reporter gene assay was performed in HEK293 cells. We used a reporter construct incorporating multimeric Lef/Tcf promoter sequences upstream of a luciferase reporter (TOPFLASH) and a control construct containing mutated promoter sequences (FOPFLASH, ref. 21). We cotransfected TOPFLASH or FOPFLASH, ?-catenin, and human wild-type Tcf-4 wild-type expression constructs into 293 cells. Optimized luciferase assays showed a basal level of TOP/FOP activity of between 4:1 and 20:1. Forty-eight hours after transfection, serum-starved 293 cells were treated with IGF-1 or insulin for 3 or 6 h. These factors alone failed to alter basal reporter activity (Fig. 6 b). A 16-h pretreatment of the transfectants with LiCl had a dramatic effect on reporter activity, with significant enhancement at 50 mM LiCl (P < 0.01 by Tukey's test for comparison with activity in the absence of LiCl). We saw a further increase in reporter activity when we added 50 ng/ml (6.5 nM) IGF-1 to transfectants pretreated with LiCl at 10 mM (P < 0.01 after 6 h of IGF-1 treatment) and at 50 mM (P < 0.01 after 3 h of IGF-1 treatment and P < 0.001 after 6 h)." provenance.
- _5 wasQuotedFrom 11035789 provenance.