Fig. 4.

Knockdown of E6AP expression stimulates estradiol-induced transcription of the Arc gene. SH-SY5Y cells (par) and SH-SY5Y cells, in which endogenous E6AP expression is stably down-regulated by RNA interference (shE6AP), were treated with 10 nM estradiol (E2), and cells were harvested at the times indicated. (A) Ten percent of the cells were used to determine levels of E6AP (α-E6AP) and, as loading control, tubulin (α-Tub). (B) The remaining cells were used to isolate total RNA followed by cDNA synthesis and determination of relative Arc mRNA levels by real-time PCR. Simultaneous determination of Actin mRNA levels served as reference. The ratio between Arc mRNA and Actin mRNA at time 0 was set to 1. Real-time PCR was performed in four independent experiments with similar results, but the time course of estradiol-induced transcription of the Arc gene varied slightly between individual experiments. The graph shows a representative example. (C) Summary of the data obtained in four independent experiments. The maximum level of Arc mRNA observed upon addition of estradiol to parental cells (e.g., mRNA level at 1.5 h in B) was set to “1” for each individual experiment and the relative increase of Arc mRNA levels observed in E6AP knockdown cells (shE6AP) was correlated accordingly. SD (± 0.27) is indicated. P value (independent two-sample t test) is depicted.