Figure 4. E1AF involved in the mouse FAK gene transcription activation in B16F1 cells.

(A) Activation of FAK promoter by ETS transcription factor E1AF. The B16F1 cells were transfected with 1 µg plasmids expressing ETS-1, ETS-2, E1AF or mock plasmid along with 1 µg p-842/+43-luc and Renilla expressing plasmid. The luciferase activity of each sample was normalized to the internal Renilla control. Then the normalized luciferase activity was standardized to that of p-842/+43-luc with vector alone. Each value is the mean ±S.D. of at least three independent experiments. (B) Mapping the regions of the FAK promoter necessary for E1AF responsiveness. The B16F1 cells were transfected with p-842/+43-luc construct or the truncated FAK promoter constructs shown above and with or without E1AF expression vector. Luciferase activity was normalized to Renilla luciferase activity and standardized to the normalized activity from p-842/+43-luc with control vector alone. Data shown are the means ±S.D. of at least three independent experiments. (C) Site-directed mutation analysis of the FAK promoter. Cells were transfected with 2 µg p-170/+43-luc, p-55/+43-luc, or p-170/+43-luc devoid of E1AF binding site (p-170/+43 m) along with Renilla expressing plasmid. FAK promoter activity in B16F1 cells was decreased in the absence of E1AF binding site. (D) Stimulatory effect of E1AF overexpression on FAK promoter activation was prevented in the absence of E1AF binding site. B16F1 cells were co-transfected with 1 µg E1AF expressing plasmid, or empty vector along with 1 µg wide type p-170/+43-luc or mutated p-170/+43 m-luc and Renilla expressing plasmid. Luciferase activity of B16F1 cells co-transfected with empty vector and wide type p-170-luc was arbitrarily set at 1. E. EMSA was performed using nuclear proteins of B16F10 cells and the ³²P-labeled mouse FAK promoter E1AF element probe. F. EMSA of the same amounts nuclear extracts from B16F10 cells and B16F1 cells incubated with ³²P -labeled E1AF element probe and supershift by PEA3 antibody. The arrows indicate the supershifted bands.