Figure 9
Functional role of CREB in PACAP-induced PRL promoter activity. (A) Effect of oPACAP38 on CREB phosphorylation and total production in grass carp pituitary cells. Pituitary cells were treated with oPACAP38 with the indicated doses or Forskolin (1 μM) for 24 hrs. After drug treatment, cell lysate was harvested for Western blot analysis on pCREB and tCREB. β-actin was blotted as internal control. (B) Statistical charts of the western blot results quantified by Image J software. (C) αT3-1 cells were co-transfected with pPRL(−1156).LUC and increasing doses of grass carp CREB expression vector CREB-pcDNA3.1 for 24 hrs. (D) αT3-1 cells transfected with pPRL(−1156).LUC were treated by oPACAP38 (10 nM, 24 hrs) in the presence or absence of CREB-pcDNA3.1 over-expression. (E) αT3-1 cells transfected with pPRL(−1156).LUC were treated by oPACAP38 (10 nM, 24 hrs) in the presence or absence of siRNA for CREB or siRNA control. (F) αT3-1 cells transfected with pPRL(−1156).LUC were treated by oPACAP38 (10 nM, 24 hrs) in the presence or absence of dominant negative CREB mutant kCREB overexpression. For transfection experiments, after drug treatment, cell lysate was prepared for dual-luciferase measurement. Data presented were expressed as percentage of control by conversing the ratio of firefly and renilla luciferase in the same sample. Data presented are expressed as mean ± SEM (n = 4) and different letters denote a significant difference at p < 0.05 (ANOVA followed by Fisher’s LSD Test).
