Rasd1 Modulates the Coactivator Function of NonO Figure 5. Rasd1 and NonO interact at the CRE-site of the target promoter to repress the transcription of endogenous cAMP target genes, NR4A1 and NR4A2, but not FOS. (A) Quantitative real-time study was performed to study the effect of over-expression of Rasd1 and NonO on endogenous CREB-target genes in HEK293T cells. Induction of the cAMP pathway with forskolin (20 mM for 45 minutes) leads to the up-regulation of cAMP target genes – NR4A1, NR4A2 and FOS (compare the respective Bars I and II of each gene). Co-transfection of plasmids expressing Rasd1 and NonO in forskolin-induced HEK293T cells leads to the down-regulation of NR4A1 and NR4A2 transcripts (compare the respective Bars II and IV of each gene). The expression of FOS transcript was not affected by the presence of Rasd1 (compare Bars II and IV of FOS). Expression of YWHAH transcript was not affected either by treatment of cells with forskolin or in the presence of Rasd1 and NonO, and was shown as a negative control (Bars I–IV of YWHAH). b-actin was used as an internal control for normalization. On the right are representative western blots showing expression of transfected NonO and Rasd1 in HEK293T cells. Actin serves as a loading control. NonO is detected by mouse anti-V5; Rasd1 is detected with mouse anti-GST; and actin is detected with anti-actin. (B) Quantitative real-time study of the effect of Rasd1 and NonO on endogenous genes of NONO-knockdown PLoS ONE | www.plosone.org 11 September 2011 | Volume 6 | Issue 9 | e24401