Regulated protein and mRNA levels of ETA receptor, ETB receptor in response to alteration of c-Jun, C/EBPβ expression.
A. Western blot analysis was used to detect the protein level of ETB receptor in HNPE cells following overexpression of c-Jun or C/EBPβ. ETB receptor protein was upregulated in membrane fractions of HNPE cell when either c-Jun or C/EBPβ was overexpressed. Calnexin, a housekeeping gene, served as a loading control. B. Relative mRNA expression of ETA and ETB receptors was analyzed using real-time PCR in HNPE transfected with c-Jun or C/EBPβ plasmid. Overexpression of c-Jun or C/EBPβ triggered an increase of ETA and ETB receptor mRNA level. Results showed as mean ± SEM, n = 3. C. siRNAs were employed to knock down c-Jun or C/EBPβ. Total RNA was extracted from HNPE cells transfected with c-Jun or C/EBPβ siRNA and 1 µg of total RNA was transcribed to cDNA. Cyclophilin A served as an internal control for normalization of equal loading of mRNA in the RT-PCR analysis. Knock-down of c-Jun or C/EBPβ significantly reduced the mRNA level of ETA and ETB receptor by more than 5 fold. Knock-down effect in protein level of c-Jun siRNA was also confirmed by Western blot, whereas knock-down effect of C/EBPβ was not detectable.