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Figure 3 | BMC Molecular Biology

Figure 3

From: Rasd1 interacts with Ear2 (Nr2f6) to regulate renin transcription

Figure 3

Transcriptional repression of renin by Ear2 can be modulated by alternating the endogenous level of Rasd1. (A) Real-time RT-PCR was conducted to determine level of endogenous renin transcription when Rasd1 was overexpressed. As4.1 cells were transiently transfected with pxJGST (2.0 μg) and pcDNA4.0 (3.5 μg) (bar I), or pGST-Ear2 (2.0 μg) and pcDNA4.0 (3.5 μg) (bar II), or pHisHA-Rasd1 (3.5 μg) and pcDNA4.0 (3.5 μg) (bar III). Representative immunoblots demonstrated Ear2 and Rasd1 transgene expression 48 hours after transfection (lower panels). (B) Semi-quantitative RT-PCR showed Rasd1 knockdown in the As4.1 cells. As4.1 cells were transfected with Rasd1 shRNA or control shRNA (4.0 μg). Cells were selected with puromycin 24 hours post transfection and harvested 48 hours post transfection. The cDNA amplified were run on a 1.5% agarose gel with G3PDH as an internal control (upper panels). A representative immunoblot demonstrated endogenous Rasd1 protein knockdown by Rasd1 shRNA (lower panel). Proteins were detected with anti-Rasd1 and anti-actin antibodies. Quantification of the immunoblots by densitometer revealed that Rasd1 protein levels, normalized against actin protein levels, were knocked down by more than 48% (lower panels). (C) Luciferase study showed that dexamethasone treatment counteracts the effect of Rasd1 shRNA knockdown. As4.1 cells were transfected with p4.1-Luc (2.0 μg), pGST-Ear2 (1.5 μg), pHisHA-Rasd1 (1.5 μg), Rasd1 shRNA or control shRNA as indicated (4.0 μg), together with pSV-β-gal (0.5 μg). Controls were transfected with pGL3-basic (2.0 μg), pSV-β-gal (0.5 μg) and appropriate amounts of the respective carrier vectors. Cell were selected with puromycin (2 μg/ml) and induced by dexamethasone (100 nM) 24 hours after transfection. Cells were harvested and analyzed for luciferase activity 48 hours after transfection. Relative luciferase activity was normalized against β-gal activity. * and ** denotes p < 0.05 and p < 0.01, respectively.

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