Figure 6

(A-C) Effects of wild-type and Rasd1 mutants on Ear2-mediated renin transcription. Luciferase studies showed that: A, COS-7; B, As4.1; C, Neuro2a. Rasd1[A178V] mutant alleviated Ear2-mediated repression of renin transcription in a magnitude similar to wild-type Rasd1 (compare bars IV and V), whereas Rasd1[G81A], Rasd1[T38N] and Rasd1[ΔCAAX] did not significantly alleviate Ear2-mediated repression of renin transcription (bars VI-VIII). (A, B and C, lower panels) Western blots showed similar expression efficiency of proteins. In all blots, expression of actin is shown as a protein loading control. * and ** denotes p < 0.05 and p < 0.01, respectively. (D) Co-precipitation followed by immunoblot showed that Rasd1[G81A], Rasd1[T38N] and Rasd1[ΔCAAX] mutations interfere with their ability to interact with Ear2. COS-7 cells were co-transfected with pGST-Ear2 and the respective Rasd1 construct expressing plasmids or the empty vector. Precipitation of GST-Ear2 with GSH-linked magnetic beads co-precipitated with Rasd1 proteins expressed from each construct (panel a, lanes I-V). However, the amount of HAHis-Rasd1[G81A], HAHis-Rasd1[T38N] and HAHisRasd1[ΔCAAX] co-precipitated were significantly less than that of HAHis-Rasd1 and HAHis-Rasd1[A178V] (compare panel a, lanes I and II with lanes III-V). Similarly, co-precipitation assays using magnetic Ni-NTA beads revealed that the amount of GST-Ear2 co-precipitated with HAHis-Rasd1[G81A], HAHis-Rasd1[T38N] and HAHisRasd1[ΔCAAX] were significantly less than that of HAHis-Rasd1 and HAHis-Rasd1[A178V] (compare panel c, lanes I and II with lanes III-V).