Fine mapping the UP site using point mutants. (a) An shRNA vector knocks down GABP alpha. HeLa cells were transfected with an empty shRNA vector (H1–2) or vectors directed against the luciferase gene (shLUC) or the GABP alpha gene (shGABP) and protein lysates collected and analysed by western blot. Control antibodies against Sp1 (Anti-Sp1) or antibodies recognizing GABP alpha (Anti-GABP) were used to detect these proteins. (b,c) The mGA and mUS point mutations alone (L6-mGA, L6-mUS) and in combination (L6-mGAmUS) were incorporated into the L6 or L6 with the RIBS site deleted (DR) promoters. These were then cotransfected into MCF-7 (b) or T47D (c) cell lines with the empty H1–2 vector or with the GABP alpha shRNA vector (+shGABP). Cells were harvested 48 hours post-transfection and analysed for luciferase activity. The data presented are the mean values ± standard deviation of a representative experiment performed in triplicate and normalized as described in Figure 2. Statistical significance was determined using a t-test and significant results (p = 0.05) are indicated by asterisks and are in relation to the L6 or L6DR vector in each group.