Collectively, BTK phosphorylation these results indicate that mTOR activation could suppress LHBs expression through a negative feedback loop that repressed pre-S1 promoter activity. To determine the critical elements responsible for pre-S1 promoter repression by mTOR signal, we constructed three 5′-deletion mutants of pre-S1 promoter reporter plasmids (Fig. 5Aa,b,c). As shown in Fig. 5Ba,b,c, luciferase activities of all these deletion mutants consistently showed that only approximately 50% of the activities were detected in pre-S2 mutant-expressed cells, compared with those in control cells, indicating that nucleotide 2789-2845 of the pre-S1 promoter was the minimal

region for mediating mTOR signal-induced transcriptional repression. A computer search further revealed that this region contains three putative transcription factor binding sites at nucleotide AZD2014 2794-2801 (site 1), 2812-2816 (site 2), and 2820-2825 (site 3) of the pre-S1 promoter. We, therefore, generated pre-S1 promoter constructs with mutations at each one of these three sites (Fig. 5Ad,e,f) for further assays. As shown in Fig. 5Bd,f, activities of pre-S1 promoter-carrying mutations at site 1 or site 3 still showed mTOR activation-dependent repression, indicating that these

two sites were not responsive elements for the mTOR signal. Interestingly and unexpectedly, the mutation at site 2 resulted in a relatively low or insignificant luciferase activity of the pre-S1 promoter (Fig. 5Be), suggesting that site 2 was transcriptionally necessary for pre-S1 promoter activity. To further evaluate the role of site 2 in pre-S1 promoter repression by mTOR, we created

one additional mutant construct, in which site 2 remains intact, but site 1 and site 3 are mutated (Fig. 5Ag). We observed that this mutant construct could negatively respond to mTOR activation (Fig. 5Bg). Therefore, the 2812-2816 site appeared to be not only transcriptionally important for pre-S1 promoter activity, but also capable of mediating a suppressive effect under the status of mTOR activation. Because sequence analysis revealed that the 2812-2816 site of pre-S1 promoter is the putative binding site for transcription factor YY1, we performed EMSA and DAPA to confirm Anacetrapib the binding of YY1 to this site. In the EMSA experiment, two major shifted bands (a and b) were detected using WT probes containing the 2812-2816 site, one (a band) of which was not detected using the Mut probes, which destroyed the 2812-2816 site (Fig. 6A). Coincubation with competitors abrogated the formation of these two bands. The data suggest that the slower migrating band (a), but not the faster migrating band (b), represented the YY1-probe complex. To further confirm this speculation, we additionally added YY1 antibody to the reaction mixtures and observed specifically reduced intensity of the slower migrating band (a). This data further verified the interaction of YY1 with the 2812-2816 site.