Functional characterization of +2.9HS. (A-B) Enhancer function of the +2.9HS. The sense (+2.9HS/s) and antisense (+2.9HS/as) sequences of +2.9HS were coupled to either the SV40P (A) or the hEPCR PP (B). Normalized reporter gene activity obtained following transfection of constructs into EA.hy926 (▦) and HepG2 (▪) cells are presented, ± SEM (n = 6). (C) Promoter function of +2.9HS/as compared with that of PP in EA.hy926 and HepG2 cells. +2.9HS/as and PP were cloned upstream of a reporter gene in a construct without any other promoter. Normalized reporter gene activity is presented relative to that of PP in EA.hy926 cells, ± SEM (n = 6). (D) Detection of antisense hEPCR transcripts. RT-PCR was performed on total RNA from EA.hy926 or HepG2 cells using strand-specific primers that specifically amplified transcripts spanning hEPCR exons 1 and 2. Nested PCR and electrophoresis was used to visualize amplified sequences. The identity of the amplified RNA transcripts (sense and antisense) was confirmed by sequencing.