Fig. 4.

4. Deletions and mutation of GRE and AF sites in Pck1 promoter. A series of constructs were made that contained serial deletions of the Pck1 promoter-firefly luciferase or contained mutations of GRE and AF sites (represented by X) and transfected into HC11 cells. Cells were incubated with the lactogenic hormones dexamethasone, insulin, and prolactin (DIP) or lactogenic hormones minus prolactin [dexamethasone and insulin (DI)] as described in Experimental Procedures, and relative luciferase activity was measured in cell extracts. A: For serial deletion analysis, portions of the Pck1 promoter (−490 bp to +72 bp, −402 bp to +72 bp, −206 bp to +72 bp) were cloned into pGL3-basic promoter vector and analyzed, or (B) −490 bp to +72 bp of Pck1 promoter was cloned into pGL3 basic vector with single site mutations of GRE and/or AF sites or (C) multiple site mutations of GRE and/or AF sites as described in Experimental Procedures. The data are expressed as the means ± SEM, n = 6; *P < 0.05.