Figure 4
TSC1 is a direct target of let-7g. (A) 293 T cells were co-transfected with pMIR-REPORT luciferase construct containing the 3′UTR of TSC1 and different doses of the let-7g mimic (10, 20 or 40 nM). Relative luciferase activity was measured after 48 h of transfection. (B) Luciferase activity of the mutant 3′UTR of TSC1 was not altered in presence of mimic let-7g (40 nM) compared to the wild-type 3′UTR. (C) let-7g transfected HT29 cell lysates were immunoprecipitated with Ago2 specific monoclonal antibody or IgG2a isotype control. RNA was isolated from immunoprecipitates using RNeasy kit. let-7g bound TSC1 mRNA expression was analyzed by qRT-PCR (left panel). Expression of Ago2 and TSC1 were checked in each set of input sample lysates by immunoblotting (right panel). Relative expression was determined after normalization to control cells. (D) Relative expression of let-7g from Ago2 immunoprecipitates was analyzed by qRT-PCR. The results are shown as mean and standard deviation from representative of three technical replicates.
