Figure 2

Rasd1 counteracts repression of renin transcription by Ear2. (A) Luciferase assay showed that transcription of renin is repressed by Ear2. COS-7 cells were transfected with pGL3-basic (2.0 μg) or p4.1-Luc (2.0 μg) or p4.1-Luc (2.0 μg) and pGST-Ear2 (1.5 μg), and with pSV-β-gal (0.5 μg). p4.1-Luc construct was generated by cloning the 4.1 kb renin promoter into pGL3-basic vector. Relative luciferase activity was normalized against β-gal activity. (B) Rasd1 alleviates Ear2-mediated repression of renin transcription in a dosage dependent manner. COS-7 cells were transfected with a constant amount of p4.1-Luc (2.0 μg), pSV-β-gal (0.5 μg) and pGST-Ear2 (1.5 μg), and with an increasing amount of pHisHA-Rasd1 (0.1, 0.2, 0.5, 1.0 and 1.5 μg). (C) Ear2 attenuates the renin promoter activity induced by retinoic acid in a dosage dependent manner. COS-7 cells were transfected with a constant amount of p4.1-Luc (2.0 μg), pSV-β-gal (0.5 μg) and pHisHA-Rasd1 (1.5 μg), and with an increasing amount of pGST-Ear2 (0.5, 1.0 and 1.5 μg). Renin transcription was induced by all-trans retinoic acid 24 hours post transfection. (D and E) Rasd1 alleviates Ear2-mediated repression of retinoic acid induced renin transcription in a dosage dependent manner. The effect of Rasd1 and Ear2 on retinoic induced renin transcription was tested by measuring the luciferase activity in varying amounts of either Ear2 (D) or Rasd1 (E). The amounts of pGST-Ear2 transfected in D were 0.5, 1.0 and 1.5 μg; the amounts of pHisHA-Rasd1 transfected in E were 0.2, 0.5, 1.0 and 2.0 μg. * and ** denotes p < 0.05 and p < 0.01 respectively. (A-E) In all luciferase assays, controls were transfected with pGL3-basic (2.0 μg), pSV-β-gal (0.5 μg) and appropriate amounts of the respective carrier vectors.