Figure 7. Two hundred and fifty-seven nt-long enhancer element is enough to restore activity at the PAS4. (A) Schematic representation of the chimeric minigene constructs: A1-A4 (1), A4 (2), A1(260-328)-A4 (3), A1(215-380)-A4 (4), A1(215-471)-A4 (5); (B) northern blot analysis of total RNA prepared from HeLa cells previously transfected with the constructs shown in (A); the α globin fragment used as a probe for the northern blots is indicated in Figure 1; the p-EGFP-C2 plasmid was used for the normalization of the transfection efficiency; the positions of the 28S and 18S rRNAs are indicated on the right; (C) the expected sizes of the mRNAs of the constructs shown in panel A up to the predicted PAS4, without pA tail (indicated by an asterisk). (D) The relative mRNA levels calculated as the ratio between analyzed mRNAs and GFP mRNA control represented in (B); quantification of the northern blot signals has been performed as described above; the mean +/− SD of four independent experiments is shown; data were analyzed using one-way ANOVA and Tukey's multiple comparison test; *** p < 0.001, ns, not significant.