Analysing the transcription start sites of Brn-3b promoter. (a) Analysis of Brn-3b promoter (BSX) activity following the mutation of key sites to identify the transcriptional start site. Wild-type (WT) promoter in row 1 (right) is represented as 100%, and all mutations are expressed as percentages of the WT promoter. Schematic shows positions of mutations at key sites (indicated by x, left), for example, upstream initiator alone (row 2, right) or in combination with other sites (rows 7 to 9, right). The proximal -278TATA mutation is shown alone (row 3, right) or in combination (rows 7 and 9, right). The effects of mutation of different intronic TA alone (rows 4 to 6, right) or in combination (rows 8 to 10, right) are also shown. Values are equalised to internal control (Renilla luciferase), and the results represent data from three independent experiments expressed as means + SD. * is used to show statistical significance between different mutant promoter constructs and wild-type constructs. (b) Western blot analysis of Brn-3b protein expression in cellular extracts prepared from MCF7 cells transfected with BSXE1E expression constructs in which Brn-3b promoter drives expression of the Brn-3b gene. WT promoter activity is shown in left column. Middle column (Δ-278TATA shows the reduction in Brn-3b protein from expression constructs containing a mutation within the proximal -278TATA promoter of an otherwise, intact construct. Right column shows untransfected control cells with no reporter and therefore represents levels of endogenous Brn-3b protein in these cells.