Figure 4

Analysis of PU-boxes. (A) Transfection analysis of the constructs -184 and -598, and their respective PU-box 1 and 2 mutants into mtCC cells. Mutations introduced into PU-box 1 and 2 are shown at the bottom. Relative luciferase activity is shown as the mean ± SD based on the activity of construct -598 without (w/o) mutation as 100 from three independent experiments, each carried out in duplicate. (B) EMSA analysis of PU-box 1 (PU1) and 2 (PU2). EMSA was carried out using oligonucleotide containing PU1 or 2 binding site as a probe (shown at the bottom) and nuclear extracts prepared from mtCC cells. Mutation was introduced into each binding site (mPU1, mPU2 in bold italics with underline) and was used as a competitor. Both PU1 and PU2 bound the same protein as shown by an arrow. (C) Transfection analysis of -598, and -598 PU-box 1 and 2 mutants into COS-1 cells with and without NF-Y. (D) Cotransfection analysis of construct -598 and PU.1/Spi-1 expression plasmid into COS-1 cells in the presence and absence of NF-Y. Relative luciferase activity is shown as the mean ± SD based on the activity of the construct -598 without NF-Y as 1 from three independent experiments, each carried out in duplicate.